Notebook

Parts Guide

In the cloning section of our notebook, our sequences are each assigned a letter code followed by a symbol. The letter codes denote the identity of sequences as follows:

A - pLacI-LasR; pLas-RBS-T4Holin
B - pLacI-LasR; pLas-RBS-sfGFP
C - pLsr-RBS-sfGFP (BBa_K1659501)
D - pLsr-RBS-T4Holin (BBa_K1659601)
E - DsbA-DNase (BBa_K1659301)
F - YebF-DspB
G - DspB (BBa_K1659200, BBa_K1659210)
H - MccS (BBa_K1659100)
I - Fla-DspB
J - DsbA-DspB (BBa_K1659201, BBa_K1659211 )
K - DsbA-Art175 (BBa_K1659002)
L - YebF-Art175 (BBa_K1659003)
M - ArtE (BBa_K1659088)
N - Fla-Art175 (BBa_K1659001)
O - Art175 (BBa_K1659000)
P - DNase (BBa_K1659300)

The symbols following the letter codes denote the type of plasmid which the sequence was being cloned into:

* - pSB1C3
# - pBAD33 for sequences A, B, C, and D; pBADHisB for all other sequences

Cloning

Week 1

Day 1

Preparation of Stock Solutions

1. gBlocks

The gBlocks ordered from IDT arrived in the form of vials of 200µg solid DNA powder.

(refer to BioBricks page for information on DNA sequences)

The gBlocks were made into 10ng/µl stock solutions in Milli-Q water for storage:

mass/ng	conc/ngµl^-1	final volumeµl
200	10	20

2. Primers

The forward and reverse primers ordered from IDT came in 32.4nmol and 34.3nmol of solid respectively.

Sequences

Forward - CTTTTTTGCCGGACTGC
Reverse - ATGATTTCTGGAATTCGC

The primers were made into 100µM stock solutions in Milli-Q water for storage:

amt/10^-9mol	conc/10^-6M	final volume/10^-6L
32.4	100	324
34.3	100	343

Preparation of Reaction Solutions

1. gBlocks

2µl of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20µl to make 1ng/µl^-1

2. Primers

2µl of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20µl to make 10µM reaction solutions. (The solutions are labelled as "Prefix primer" and "suffix primer" in eppendorf tubes in the fridge.)

Polymerase Chain Reaction

25µl reactions were run according to the PCR protocol here

* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s online tool here.

** Add components in order of decreasing volume for maximum ease-of-pipetting.

*** When reaction mixture is being made up, all components as well as the mixture itself are to be kept on ice, as the Master Mix is a high-fidelity polymerase that will recognize the primers as being incorrectly base-paired and be able to hydrolyse the primers if kept at room temperature.

**** Use Q5 enzyme in the cold room to avoid defrosting and freezing the original stock of Q5 enzyme. This could decrease the activity of Q5 enzyme. Bring ice bucket to the cold room to bring Q5 into the bench.

***** Make sure that the primer and small amounts of DNA and primer doesn’t stick onto the side of the tube or the tip.

The reaction mixture tubes were positioned in an Eppendorf Mastercycler nexus X2 and the PCR program was run.

* DNA denaturation can be performed at 98℃ because of the high thermal stability of the Q5 polymerase

** A PCR takes 20-30 seconds to extend a sequence by 1kb, and since our longest sequence is ~2kb the extension time was determined to be 60s per cycle.

Gel Electrophoresis of PCR-Amplified gBlocks

An agarose gel was prepared according to the agarose prepartion protocol.

DNA preparation:

The contents of the PCR tubes need to be stained with a loading dye to help visualize its migration.

To each 25µl of content in each PCR tube, 10µl of blue loading dye was added.

Day 2

Gel Electrophoresis of PCR-Amplified gBlocks (continued from 22/06)

gBlock sizes for reference

Lane 1: 10µl of 2-Log Ladder

Lanes 2-15: 20µl of DNA and loading dye mixture prepared on 22/06

A potential difference of 120V was applied across the electrodes (the higher the voltage, the faster the gel will run but the poor the separation will be; DNA separation is typically done in the 120-140V range) for 80 minutes. As long as DNA has passed ⅔ of the column or purple dye have passed purple area of the gel, the gel is ready to get into the EtBr.

The gel was then stained and the bands were visualized; protocol

Results

PCR Results

The 7 bands corresponding to expected DNA sizes were excised using a razor blade for cleaning and extraction. The other sequences, along with J which only showed a very weak band, will be re-PCRed under modified conditions at a later date.

The excised gel chunks were transferred to centrifugation tubes.

Extraction of DNA (PCR product) from Agarose Gel

The extraction was performed using the E.Z.N.A. Gel Extraction Kit made by Omega Biotek, according to the Spin Protocol.

The excised agarose chunks needed to be dissolved in a minimum of 1mL of XP2 Binding Buffer per gram of gel. The heaviest chunk of gel weighed 0.16g and as such 160µl of buffer was added to each tube.

* As the tubes were spun with their lids open, they were placed such that lids pointed away from the direction of spinning.

Restriction Digest of Extracted PCR product

Restriction digest was performed using EcoRI-HF (5’) and SpeI (3’) restriction enzymes.

NEB’s Double Digest Finder was invoked and it was determined that CutSmart Buffer would be used for the digest.

The buffer was completely defrosted and shaken before use.

There is a recommended digestion protocol on NEB Cloner. 50µL reaction mixtures were set up with component volumes as recommended, except for the DNA where all 30µL of the extraction mixture (in elution buffer) were used in the digest. There would be up to 50ng of DNA in each tube of extraction mixture (from the gel bands).

Incubation at 37℃ was also done for 2 hours (ThermoMixer program 3) instead of the recommended 5-15 minutes, with shaking at 300rpm.

Restriction Enzyme Digest of the iGEM HQ linearised pSB-1c3

125ng of pSB-1C3 was dissolved in 5mL Milli-Q water.

The digest was performed using the modified NEBCloner protocol.

* We did not add phosphatase because it is assumed that with different sticky ends the vector cannot religate

DNA ‘Cleanup’ using EZNA enzymatic reaction kit

Upon completion of restriction digest incubation, the gBlocks and the plasmid backbones were purified again using the E.Z.N.A. Gel Extraction Kit. PCR products and plasmid backbones need to be purified in order to remove remaining impurities including agarose gel and restriction enzymes. At the elution step, the gBlock inserts were eluted using 30µL elution buffer whereas the plasmid backbone was eluted using 50µL of it.

DNA Quantification using NanoDrop

1µL water and tissue were first used to clean the stage. A blank reading was made using 1µL of elution buffer, and 1µL of each sample was measured for concentration:

Sample	Concentration/ngµl
C	11.3
D	3.5
E	0.8
H	0.9
J	1.6
L	7.7
N	3.6
pSB-1C3	1.6

Ligation of gBlock Sequences with pSB-1C3 Backbone

Ligation was completed; protocol

Preparation of Competent E. coli Cells

A sample of E. coli DH5ɑ stored at -80℃ was defrosted and inoculated in 5mL of LB in a 125mL conical flask (volume of LB 10% of flask volume so as to achieve sufficient aeration). The culture was left to grow overnight at 37℃.

Re-PCR of DNA Sequences

Two sets of sequences A,B,F,G, I, J, K, and M were PCRed using the same protocol as 22/06, with one set using a 55℃ annealing temperature and another using 50℃ annealing temperature.

Day 3

Re-PCR of DNA Sequences

Refer to protocol from 1.0

PCR Results

The amplified sequences A, B, F, G, I, K, J, and M were loaded with blue dye and run on agarose gel as per standard protocol

Sequences G and J showed bands corresponding to the expected sizes at both 50℃ and 55℃ annealing temperatures.

The bands were excised and extracted according to standard protocol (E.Z.N.A. Gel Extraction).

Plasmid recovery

The concentration of pSB1C3 plasmid recovered on 23/06 according to the NanoDrop was low (1.6ng/µl). To obtain more plasmids, we restriction digested a construct made by last year’s, pSB1C3 + abijk, to recover the pSB-1C3 content from the construct.

Set up the following reaction mixture:

Component	Volume/µl
DNA (pSB1C3 + insert)	5
EcoR1-HF	1
SpeI	1
Cutsmart buffer	2
Water	11

During the incubation set up 1% agarose gel by putting 1g powder agarose in 100ml; 1% gel is more efficient at separating larger fragments

Results

Incubate the mix above for 2hrs at 37℃
Incubate for 30mins at 95℃ to inactivate restriction enzymes (Inactivation temperature of SpeI is 80℃, and EcoR1-HF is 65℃)
Cool and spin (there will be condensation at the top of the eppendorf)
Add 1µl CIP (phosphatase) and incubate for 30mins at 37℃
Add loading dye
Load each DNA on the gel
Stain in EtBr for 30 + 20 mins (staining not very clear after 30 mins)

Extraction of “sticky” pSB1C3 (recovered from 2014 pSB1C3+abijk) from Gel

E.Z.N.A. Gel Extraction

Gel weight:

0.53g ⇒ 0.53mL Binding Buffer

30µL of elution buffer used

Repeat PCR with higher DNA concentration (for the sequences that previously did not yield clear bands)

Sequences A, B, F, G, I, J, K and M have gone through a second round of PCR as they previously failed to yield clear enough bands, and ethidium bromide staining shows that G and J have been successfully isolated as a result.

A, B, F, I, K, L and M still have not had successful PCR runs, as such an alternative PCR protocol with higher DNA concentration was attempted on them instead:

Reagent	Volume/µl
gBlock Template (1ng/µl)	5
Forward Primer	1.25
Reverse Primer	1.25

* A total of 5ng gBlock as opposed to 1ng at 58℃ annealing temperature present in reaction mixture; rest of the protocol is same as prescribed

Results

Alternative protocol does not work.

Gel extraction of G and J

Standard E.Z.N.A. Gel Extraction protocol followed.

Gel weights:

J - 0.34g ⇒ 0.4mL Binding Buffer

G - 0.3g ⇒ 0.4mL Binding Buffer

30µL of elution buffer used.

Restriction Digest of G and J

A restriction digest was then performed on the extracted DNA.

NanoDrop Quantification

J: 2.7ng/µl
G: 1.7 ng/µl
pSB-1C3: 1.4ng/ul - there is 8-9ul of this left in the freezer in drawer 4, labelled with Psb1c3 EcoRI, SpeI and 24/06/2015

Ligation

Ligation perform according to protocol.

Transforming E. coli cells with Plasmid DNA

Competent e.coli cells were first prepared, according to the protocol here.

Sample C,D,E,H,J,L and N to be transformed, according to the protocol here.

Day 4

Growth and Culture of Bacteria

For the protocol please click here.

Plates incubated overnight show that vectors corresponding to C,D,E,H,J,L and N were taken up [however, later we find that ligation had failed, and so gBlocks C,D,E,H,J,L and N were never inserted]. There are at least 5-7 colonies for each biobrick.

Choose three colonies from each plate. The colony should not be too small or too large and should be reasonably spaced from the others.

Separately incubate each colony in test tubes overnight.

This process would significantly increase the amount of plasmids containing biobricks that we want. Plasmids can be extracted later.

Transformation of J and G (24/06 PCR Product) into competent E. coli cells

Competent cells are already made in stocks and can be found in the -80℃ freezer: we don’t need to prepare them again. J and G comes from repeat PCR done in Day 3.

Primer Design

Since A, B, F, G, I, K, M have repeatedly failed to be PCR-amplified, longer primers were designed to replace the old primers for the PCRing of these gene sequences.

Primers that would give the sequences new restriction sites to allow their insertion into pBAD33 and pBAD/HisB expression vectors were also designed.

Day 5

Plasmid Extraction

For the plasmid extraction protocol see here.

pSB1C3 shipping vector containing gBlocks from Day 1, as well as blank pSB-1C3 shipping vector, pBAD/HisB expression plasmids, and pBAD33 expression plasmids were extracted from overnight cultures of E. coli DH5 using E.Z.N.A. Plasmid DNA Mini Kit I.

(refer to pages 10-12for Mini Kit protocol. Some special notes:

All optional steps were carried out except equilibration step.
After centrifugation in step 2, the tubes were pulsed before the excess supernatant was removed through pipetting.
Steps 6 and 7 (involving solutions II and III need to be carried out in quick succession (adhering to the short incubation time) to ensure good results. It is advisable to do these two steps in pairs as in step 6 the tubes need to be tightly capped once solution II is added.
The precipitate formed in following step 7 does not pellet well after centrifugation in step 8, and hence the suspension needs to be removed immediately to prevent resuspension.
The inversion in step 6 needs to be done gently so that genomic DNA of the bacteria are not extracted along with the desired plasmid DNA.

Plasmid Quantification

1µl of the each of the extracted plasmids were dropped onto the NanoDrop machine for concentration quantification. The results are as below:

DNA	c/ngμl^-1	DNA	c/ngμl^-1
pBAD/HisB	36.4	mccS (H1)	61.5
pBAD33	34.4	mccS (H2)	129.6
pSB1C3	142.1	DspB+DsbA (J1)	38.8
lsr + GFP (C2)	122.9	DspB+DsbA (J2)	46.3
lsr + GFP (C3)	40.3	DspB+DsbA (J3)	41.9
lsr + Holin (D1)	40.2	Art175+YebF (L1)	64.6
lsr + Holin (D2)		Art175+YebF (L2)
lsr + Holin (D3)	44.1	Art175+YebF (L3)	43.2
DNase+DsbA (E1)	77.4	Art175 + Fla (N1)	103.5
DNase+DsbA (E2)	43.4	Art175 + Fla (N2)	48.8
DNase+DsbA (E3)	40.7	Art175 + Fla (N3)	134.7

Plasmid Digest

The plasmids were digested using restriction enzymes EcoRI-HF and PstI (NEB) at 37℃ for 90 minutes. For the protocol see here.

Gel layout

10µl of blue gel loading dye was added to each tube of digested plasmids. 20µl from the contents of each tube were then loaded into a 30-well agarose gel according to the following schematic:

(*the pSB-1C3 well has pSB-1C3 with abijk insert)

The ladder well has 10µl of 2-Log Ladder added into it.

A potential difference of 120V was applied across the gel for 40 minutes before it was stained with ethidium bromide for 30 minutes.

Analysis of Results

Analysis

The first three lanes produced expected results: pBAD33 and pBAD/HisB being “blank” plasmid backbones, the pSB-1C3-abijk lane giving two bands corresponding to similar sizes (being pSB-1C3 and abijk insert respectively).

All the other lanes seem to be showing only products in the 2kb range, which corresponds roughly to the size of the pSB-1C3 backbone, but the sizes are not uniform. Nonetheless, we know that the plasmid extraction step was carried out correctly as it did yield products approximating what we were looking for.

If it was a matter of the ligation step carried out on 23/06 failing entirely, we should be expecting a uniform row of backbone bands. Instead, there are minor but noticeable size variations between each band which cannot be successfully explained by failed digestion/ligation. It is thus speculated that the pSB-1C3 stock we received from iGEM HQ had suffered from varying extents of DNA degradation such that the restriction enzyme cut sites on their ends were no longer.

Week 2

Day 6

PCR of samples C,D,E,H,J,L and N

Samples C,D,E,H,J,L and N were PCRed according to the protocol here.

* Use the Q5 HF Master Mix that has been kept at 4℃ in the cold room and not the frozen sample because repeated freeze thaw cycles are not good for the Master Mix.

** Remade 50µl 10uM primer stock from the 100uM stock solution (5µl primer in 45µl Milli-Q water)

*** Use DNA 10ng/µl stock solutions made up on Day 1

Gel electrophoresis of C,D,E,H,J,L and N

Results

Refer to gel electrophoresis protocol in section 1.1

Only J showed no clear band corresponding to the expected sequence size.

EZNA gel extraction of C,D,E,H,J,L and N

Refer to section 1.13 of the protocol guide.

Excise bands C,D,E,H,L, and N using razor blade, and the excised agarose chunks needed to be dissolved in a minimum of 1mL of XP2 Binding Buffer per gram of gel. For instance, the heaviest band was 0.27g, requiring 0.3ml Binding Buffer to each eppendorf tube.

Restriction Digest of Gel Extracted C,D,E,H,J,L and N

Refer to section 1.2 for the protocol.

Following the gel extraction spin protocol above, extracted PCR DNA needs to be ‘cleaned up’ of restriction enzyme and agarose. The protocol for this can be found from the last enzymatic protocol in EZNA gel extraction kit. This process is to be done in 30/06 (Day 7).

Recovery of pSB-1C3 vector from 2014 pSB-1C3 + insert

Component	Volume (10µl)
pSB-1C3 + insert	10
SpeI	1
EcoR1 HF	1
Cutsmart	2
Milli-Q	6

Incubate for 2hrs 37℃
Heat and inactivate the restriction enzymes at 95℃ for 20mins
Cool down in the room temperature, spin (condensation on the side of the eppendorf from heating)
Add 1µl CIP and incubate at 37℃ for 30mins
Add 5µl loading dye

* Phosphatase is added to prevent the vector religating to insert. In the case of PCR amplification of (non-plasmid) gene sequences phosphatase does not need to be added to PCR product because those two ends are unlikely to ligate onto itself.

** Must start preparing for agarose gel to run 2014 pSB1c3 + Insert. The gel will distinguish pSB1c3 vector from insert, allowing us to extract the vector from the gel and ligate with PCR product. Ligation is to be done in 30/06 (Day 7).

Results

Gel electrophoresis of pSB1C3.

EZNA gel extraction protocol on recovered pSB1c3 vector

Refer to section 1.13 for the protocol.

Start with excising the band that corresponds to the base pair length of pSB1c3 vector.

Following the gel extraction spin protocol above, extracted Vector DNA needs to be ‘cleaned up’ of restriction enzyme and agarose. The protocol for this can be found from the last enzymatic protocol in EZNA gel extraction kit. This process is to be done in Day7.

Growth and culture of E. coli transformed with 24/06 PCR Product (J and G)

Refer to section 1.6 for the protocol.

* LB has to be clear. The LB on the shelf was cloudy and therefore contaminated from last week so start with a new bottle

** Colonies were of variable size which could mean that some of the colonies are contaminated. Therefore, when picking the 6 colonies, pick 2 small, 2 medium and 2 large colonies

Day 7

From yesterday

E.Z.N.A enzymatic reaction cleanup protocol for Restriction Digest products of C, D, E, H, L, and N (1.21)

At the elution step, the gBlock inserts were eluted using 30µL elution buffer whereas the plasmid backbone was eluted using 50µL of it.

E.Z.N.A gel extraction protocol for pSB-1c3 vector isolated from 2014 pSB1C3+insert (1.13)

E.Z.N.A enzymatic reaction protocol for pSB-1C3 gel (1.21)

Ligation of 29/06 PCR products to pSB-1C3 (1.3)

Some notes:

Our component volumes were slightly different from that in day 2 due to the different amount of gBlock/vector we had. (We divided the amount of pSB-1C3 between our 6 samples)

Component	Volume/μl
Digested DNA (gBlock)	30
pSB-1C3	8
T4 DNA ligase buffer	5
T4 DNA ligase	1
MilliQ water	6

Mixtures were incubated on thermomixer at 16 ℃ for 16 hours until 8.45 a.m. on 1/7/15, taking care to vortex before placing on thermomixer.

Plasmid Extraction using miniprep kit (1.7)

Sequences G [pSB-1C3] and J [pSB-1C3] were extracted from overnight cultures of E. coli DH5𝛼 using E.Z.N.A. Plasmid DNA Mini Kit I.

refer to pages 10-12 for Mini Kit protocol. Some special notes:

All optional steps were carried out except equilibration step.
After centrifugation in step 2, the tubes were pulsed before the excess supernatant was removed through pipetting.
After addition of solution I/RNAse, vortexing/vigorous shaking of the tubes should be avoided to prevent shearing of nucleus and undesirable accidental extraction of chromosomal DNA.
After addition of solution I/RNAse, resuspension of pellet can be done by dragging the tube along an eppendorf rack.
Steps 6 and 7 (involving solutions II and III need to be carried out in quick succession (adhering to the short incubation time) to ensure good results. It is advisable to do these two steps in pairs as in step 6 the tubes need to be tightly capped once solution II is added.
After addition of solution II, the waiting time before proceeding to the next step should not be more than 5 minutes.
The precipitate formed in following addition of solution III does not pellet well after centrifugation in step 8, and hence the suspension needs to be removed immediately to prevent resuspension.
The inversion in step 6 needs to be done gently so that genomic DNA of the bacteria are not extracted along with the desired plasmid DNA.

50µL of elution buffer per tube was used at the end because plasmid DNA is being eluted.

Plasmid Quantification: Nanodrop (1.22 Nanodrops are usually done following restriction digest and transformation)

1µl of the each of the extracted plasmids were dropped onto the NanoDrop machine for concentration quantification. The results are as below:

DNA	Conc. /ngμl^-1	DNA	Conc. /ngμl^-1
J1	62.4	G1	67.4
J2	77.2	G2	81.5
J3	39.5	G3	80.2
J4	61.4	G4	88.0
J5	37.9	G5	26.3
J6	134.3	G6	95.7

Plasmid Restriction Digest for 24/06 PCR Product 1.2

* 0.5ul enzyme despite digesting a plasmid because test digest

**must refer to the master sheet each time

As many tubes were being handled, the required reagents were pre-mixed:

Reagent	Volume / μL
2.1 Buffer	50
Milli-Q	350
EcoRI-HF	12.5
PstI	12.5

3μL of each insert-containing plasmid was transferred into PCR tubes, and 17μL of the reagent mix was added to each PCR tube.

The plasmids were digested using restriction enzymes EcoRI-HF and PstI (NEB) at 37℃ for 120 minutes.

Gel Electrophoresis of Digested Plasmids from 24/06 PCR Product 1.1

Results

5µl of blue gel loading dye was added to each tube of digested plasmids.

20µl from the contents of each tube loaded. Gel run under 80V for 40 minutes:

G5, J3, and J5 were also the samples that showed irregularly low concentrations when analysed with the NanoDrop.

As such, G5, J3, and J5 were discarded while the other samples, being potentially-viable BioBricks, were freeze-stored for verification by gene sequencing at a future date.

Preparing new primer stock solutions

Primer	amt / 10^-9 mol	conc / 10^-6 M	Milli-Q vol / 10^-6 L
Posy Suffix Holin	28.7	100	287
SMAP 29 Forward	31.2	100	312
DspB Forward	27.7	100	277
Art-E Prefix	35.4	100	354
DNase Forward	18.2	100	182
YebF Forward	29.1	100	291
Art-E Suffix	19.4	100	194
Art 175 Forward	27.5	100	275
Fla Forward	26.5	100	265
Pre-prefix Holin	22.9	100	229
MccS Forward	29.2	100	292
DsbA Forward	27.0	100	270

Protocol:

Pulse spin primers
Add fresh Milli-Q as above table
Leave for 30 mins at 37C in the shaking block (allowing DNA to resuspend)
Dilute to 10µM

PCR Amplification of gBlocks Using New Primers

***Refer to the Master Table for detailing gBlock-primer combinations for both preparation for the gene sequences’ eventual transformation into pSB1C3 shipping vector as well as pBAD33 or pBAD/HisB expression vector.

Primer naming and explantion can be found here.

With reference to the Master Table, standard PCR reaction mixtures (1uL 1ng/uL DNA template, 1.25uL 10uM forward primer, 1.25uL 10uM reverse primer, 9uL Milli-Q water, 12.5uL Q5 Master Mix) was set up for A*-F*, H*, I*, K*-N*, A#-E#, K#, N#, L#, H#, M#, O#, and P#.

G* and J* were not run because we already potentially have viable BioBricks for them from the plasmid extraction done earlier today (24/06 PCR batch).

DspB-containing setups J#, I#, F#, G# were not run because DspB has a BspHI restriction site in the middle of its sequence. The DspB-containing gBlocks will first be QuickChange-PCRed as an insert in the shipping vector to remove the restriction site by introducing a point mutation to codon-swap before being redigested out of the shipping vector and ligated into the expression vector.

Some compromises had to be made in terms of annealing temperature as there were only 2 PCR machines available and hence only 4 different programs could be run in parallel. The annealing temperatures were set up as below:

Annealing T / ℃	Label
72	A-F, H, I, K-N, A#-D#
67	E#, K#, N#, P#
61	L#, H#
70	M#, O#

Reaction times and other temperatures were set up according to standard PCR protocol 1.0

Day 8

Gel Electrophoresis of 30/06 PCR Products

Protocol 1.2

Results

5uL loading dye added per PCR tube. Gel run under 120V for 45 minutes:

Sizes of each fragments can be referred to the Master Table, and the following fragments that show clear bands have been excised and sent for sequencing.

Excised: C*, D*, H*, L*,M*, N*, C#, D#, E#, H#, K#, L#, M#, N#, P#

Restriction Digest of 30/06 PCR Products

Protocol 1.2

Grouping by required restriction enzymes -

EcoRI-HF + PstI-HF: C, D, H, L, M, N

Reagent mix (7-portion) first made up:

Component	Volume / µL
CutSmart Buffer	35
Milli-Q	98
EcoRI-HF	3.5
PstI-HF	3.5

20µL of reagent mix was added to each tube containing 30uL DNA products.

BspHI + PstI-HF: E#, K#, N#, L#, H#

Reagent mix (6-portion) first made up:

Component	Volume / µL
CutSmart Buffer	30
Milli-Q	84
EcoRI-HF	3
PstI-HF	3

20µL of reagent mix was added to each tube containing 30µL DNA products.

BamHI: C#, D#

Since there are only two tubes to be handles the reagents were directly added to each tube:

Component	Volume / µL
3.1 Buffer	5
Milli-Q	14
EcoRI-HF	0.5
PstI-HF	0.5

NcoI, PstI: M#, P#

Since there are only two tubes to be handles the reagents were directly added to each tube:

Component	Volume / µL
3.1 Buffer	5
Milli-Q	14
Ncol	0.5
PstI	0.5

Incubation at 37°C for 2 hours, with 300 rpm shaking. Upon completion, samples stored at -20°C.

Plasmid Design

Quikchange plasmids for DspB were designed and ordered.

VF2 and VR plasmids, used for sequencing pSB1C3-contained inserts were also ordered.

Transformation of 29/06 ligation products (containing C, D, E, H, L, N gBlocks)

Protocol 1.5

*only 9 tubes of competent DH5alpha E. coli left in the freezer after this round of transformation, as such more will need to be made by the end of the week

Conduct plating under the filter hood. Add antibiotic to molten agar whilst in bottle, such that the antibiotic (Chl or Amp) is diluted by 1000 times. Then gently mix. Pour just enough agar to cover the surface of the petri dishes. Plates should take ~30mins to set.

Then add volume of E. coli according to protocol, using beads to spread across petri dish.

Day 9

Plasmid restriction digest - pSB1C3, pBAD33, and pBAD/HisB

Restriciton digest of SB1C3, pBAD33, and pBAD/HisB completely, using the 1.2 protocol and consulting the master table for the correct buffer and restiction enzymes.

Resultant Gel

100mL 1% agarose made up, small plate loaded and remainder left in bottle on shelf.

Total volume per tube is 35uL. Each tube’s contents split into two wells (17.5µL per well), and gel was run on 80V p.d. for 45 minutes.

* Gel can help tell if any digestion occurred at all - if no digestion occurred plasmid would still remain circular, and circular plasmid would encounter more resistance migrating through the gel matrix than linear plasmid of the same size and hence appear to be bigger than expected when compared against the ladder.

Bands were excised and the heaviest chunk was 0.66g. 670uL of XP2

Binding Buffer was added to each excised chunk to initiate E.Z.N.A. Gel Extraction protocol.

Enzymatic Reaction Clean-up - 30/06 PCR Products

Protocol is 1.13

Retrieve products (on labelled yellow rack) from -20°C and perform clean-up.

The volume of the restriction digest reaction done on 01/07 was 50µL per tube hence according to protocol 50µL of XP2 Binding Buffer added to each tube.

Upon completion of protocol, tubes placed back into -20°C until plasmids ready for ligation.

Growth and Culture of Bacteria - 29/06 PCR Products

Refer to section 1.6 of the protocol guide.

*note: new bags of inoculation loops are placed next to the 37°C incubators for agar plates

LB agar plates of C, D, E, H, L, N collected.

Three colonies selected from each plate to set up three separate overnight cultures each.

Ligation of 30/06 PCR Products(

Refer to section 1.3 of the protocol guide.

Component	Volume/µ
Digested and cleaned PCR products	30
Digested and cleaned plasmids (pSB1C3: C, D, H, L, M, N; pBAD33: C#, D#;pBAD/HisB: E#, K#, N#, L#, H#, M#, P#)	8 for pSB1C3 10 for pBAD337 for pBAD/HisB
T4 DNA Ligase	1
T4 Buffer	5
Milli-Q	6 for pSB1C3 4 for pBAD33 7 for pBAD/HisB

Incubate reaction mixture at 16°C overnight.

Preparation of Competent E. coli DH5alpha

Refer to section 1.4 of the protocol guide.

*Sterile technique used

Test tube filled with 5mL LB broth.

GW opened new bag of inoculation loops to steal a colony off one of Elaine’s streaked plates (marked 29/06) and inoculated it in the filled test tube.

Test tube left to incubate at 37°C overnight.

Day 10

Plasmid for 29/06 PCR Products

Refer to section 1.7 of the protocol guide.

Yesterday, collected LB agar plates of C, D, E, H, L, N and Interlab are incubated overnight. Selected three colonies from each of C, D, E, H, L and N, and only one for each of the Interlab colonies (we assume that the plasmids provided by iGEM HQ for Interlab were all the same).

Therefore we shall be performing EZNA plasmid extraction on 22 samples. Follow EZNA Mini Kit I Spin Protocol (pg10-12).

50µL of elution buffer per tube was used at the end because plasmid DNA is being eluted.

Also, aspirate = pipetting!

Measured the concentration of the different samples using nanodrop

Sample	Concentration (ng/μl)
C1	46.7
C2	35.6
C3	52.7
D1	43.2
D2	95.8
D3	30.4
E1	45.1
E2	112.4
E3	46.7
H1	71.6
H2	48.2
H3	48.5
L1	48.7
L2	56.2
L3	69.2
N1	57.0
N2	47.0
N3	68.3

Digest aliquots of each of the 22 samples, and run on gel. Refer to protocol 1.2

From gel, determine the degree of success of these samples.

Preparation of Competent E. coli DH5alpha

Refer to protocol 1.4

Transformation of 30/06 PCR Products (Ligated on 02/07) (Protocol in section 1.5)

Refer to protocol in section 1.5

Antibiotics:

pSB1C3, pBAD33 - Chl
for C*, D*, H*, L*, M*, N*, C#, D#
pBAD/HisB - Amp
for E#, K#, N#, L#, H#, M#, P#

Raffy, Leon: Moving of cells plated on 3/7/2015 from the incubator to the cold room. All pSB1C3 plates had reasonable colony density. A significant proportion of pBAD/HisB plated had no colonies. Plates with no colonies will be reincubated for half a day on 6/7/2015.

Week 3

Day 11

Preparation of Overnight Cultures for 30/06 PCR Products

Plate	Colony	Plate	Colony
C#	1	C#e	1
D#	0	D#e	0
E#	2	E#e	2
H#	0	H#e	3
K#	0	K#e	0
L#	0	L#e	0
M#	0	M#e	0
N#	0	N#e	0
P#	1	P#e	0

Plate	Colony	Plate	Colony
C*	Yes	C*e	Yes
D*	3	D*e	0
H*	Yes	H*e	Yes
L*	Yes	L*e	Yes
M*	0	M*e	3
N*	Yes	N*e	Yes

Colonies grew for L* and N*, but because the MiniPrep run on 03/07 already showed the correct bands for them, their colonies were not cultured overnight.

Sequence	Colonies picked
C*	3
D*	3
H*	3
M*	3
C#	2
E#	4
H#	3
P#	1

See protocol guide to find out how to make overnight cultures.

PCR amplification of A,B,D,E,F,I,K,M for pSB1C3

1. Primer used:

* primer pair (pre prefix holin and post suffix holin)

Comments:

Attempting A, B, F and I again as they have never been successfully amplified before.Attempting A, B, F and I again as they have never been successfully amplified before.

NB: F and I are DspB-containing gBlock which we eventually hope to QuikChange to get rid of the BspHI restriction site in them and put them into pBAD/HisB expression vector

2. PCR all the # sequences with the appropriate primers

Follow 1.0 PCR protocol, and primer list is in the Master Table.

Restriction digest for pSB1C3, pBAD33 and pBAD/HisB

Gel photo

Follow restriction digest protocol.

pSB1C3 and pBAD/HisB were successfully digested and eluted in 1% gel. Band for pBAD33 could not be found.

Excised bands were stored in -20℃ for cleanup tomorrow.

Primer Preparation

Preparing new primers (solution and dilution) - VF2, VR, QuikChange forward, QuikChange reverse.

First make 100uM out of the freeze dry solid:

Spin down solid
The amount of primer is given in y nmol for each tube. Add 10y µL of water to each tube to make 100µM.
Shake/vortex and mix evenly.

For VF2, VR - take 3.2ul out of the 100µM stock and dilute with 96.8µL Milli-Q to make 3.2pmol/uL sequencing primer solution.

For the QuikChange primers, dilute to 10uM as per normal.

Sequencing of BioBricks

5µl of each plasmid along with 100uL of 3.2pmol/uL sequencing primer sent to SourceBioScience for sequencing:

Sequence	Label Assigned
C 3	pSBLsrGFP3
D 2	pSBLsrHolin2
E 2	SBDNaseDsbA2
G 6	pSBDspB6
H 1	pSBMccS1
J 2	pSBDspBDsbA2
L 3	pSBArt175YebF3
N 3	SBArt175Fla3

Gel Electrophoresis of PCR Products

Correct bands were obtained for D*, C#, G#, H#, J#, K#, L#, N#. Still awaiting reply on insert length for O# and P# to determine whether the bands are correct.

Day 12

NanoDrop Analysis of Plasmids Digested on 06/07 (carried out after gel extraction)

Plasmid	Conc / nguL^-1
pSB1C3	5, 9.5
pBAD/HisB	1.8, 1.6

Since pBAD/HisB is very low in concentration, more of it will be digested.

Restriction Digest of pBAD33

Component	Volume/µL
Plasmids	10
BamHI	1
Milli-Q Water	7
3.1 Buffer	2

37℃, 2 hours (heat up another block to 95℃)
Heat inactivation, 95℃, 20 minutes
Melt, cool, and pour 1% agarose
Pulse spin
CIP dephosphorylation (1 µL CIP), 37℃, 30 minutes
Load dye and run gel

pBAD/HisB digest - 6uL water, 1µL NcoI, 1µL PstI, 2µL 3.1, 10µL plasmid

Gel extraction of digested plasmids

Plasmids digested on 6/7:

400µL of Binding Buffer added to each tube containing excised band.

After first elution, concentrations turned out to be low (see NanoDrop table at the top of this document), hence a second elution was done, again using 50µL of elution buffer.

Plasmids digested on 7/7:

340uL of Binding Buffer; one elution with 50µL.

Plasmid Extraction from 06/07 Overnight Cultures (30/06 PCR)

Follow EZNA DNA Mini Kit I Spin Protocol - eluted 50μl

freezer: expression vector ones (the ones with #) on orange rack in bottom drawer (will be dealt with tomorrow), psb vector ones (the ones with *) in white box with other psb constructs in top drawer

NanoDrop analysis:

Sequence	Conc / ngµL^-1	Sequence	Conc / ngµL^-1
C* 1	58.3	M* 3	49.2
C* 2	67.5	C# 1	64.5
C* 3	66.0	C# 2	25.1
D* 1	6.5	E# 1	37.8
D* 2	43.3	E# 2	43.1
D* 3	56.7	E# 3	30.5
H* 1	30.0	E# 4	67.3
H* 2	45.9	H# 1	36.5
H* 3	44.5	H# 2	28.8
M* 1	51.1	H# 3	37.3
M* 2	53.1	P# 1	179.2

Gel electrophoresis of Plasmid Extraction from 06/07 Overnight Cultures (30/06 PCR)

Gel Photo

Electrophoresis of 10uL plasmid with 5uL loading dye

N.B. forgot to digest - will do this on 08/07/15 (tomorrow)

Analysis of sequencing data from 06/07/15

Of the stuff sent for sequencing (03/07 miniprep):

Sample	Results	Action Point
C*3	corresponds to pSB1C3 Lsr GFP	-
D*2	Corresponds to pSB1C3 Lsr GFP (does not correspond to pSB1C3 Lsr Holin as expected - contamination in all three D* wells were already apparent in gel (see 06/07))	Wrong insert, need to redo from scratch (start from PCR again)
E*3	DsbA DNAse missing, sequencing only gives blank pSB1C3 vector	Send another miniprep product in the triplicate (E1 or E2) for sequencing.
G*6	Forward sequence good enough to confirm (~70% length of DspB sequence) that it’s pSB1C3 DspB, but reverse sequence dropped off too early for double confirmation	Ask Source Bioscience to redo VR (reverse) sequence
H*1	Corresponds to pSB1C3 MccS	-
J*2	Corresponds to DsbA DspB, but base 2964 had a C->A point mutation	Send another miniprep product in the triplicate (J1 or J3) for sequencing
L*3	Corresponds to pSB1C3 Art-175 YebF	-
N*3	Corresponds to pSB1C3 Art-175 Fla	-

Results: 4, potentially 5 BioBricks successfully made in [pSB1C3] format. Successful BioBricks to be stored in separate box in preparation for sending to Registry, creating Database page etc.

Ligation of 06/07 PCR to Appropriate Digested Plasmids

pBAD33: C#1, C#2 (one of these were wrongly digested using CutSmart instead of 3.1), D# (previously digested using wrong buffer), D#3.1

pSB1C3: D*

pBAD/HisB: E#, G#, H#, J#, K#, L#, N#, O# (previously digested using wrong buffer), O#3.1, P# (previously digested using wrong buffer), P#3.1

Refer to section of the protocol guide.

Left overnight at 16C.

Day 13

To Do

Prepare plates for transformation

Transformation of ligated plasmids

Gradient PCR:

Group A: A*, B*, F*, I*, K*
Group B: A#, B#
Group C: I#, F#

To Note

Re-sequenced VR read and DspB confirmed as correct = another biobrick.

QuikChange PCR on DspB

Standard PCR protocol 1.0

72C annealing temp
2 min extension time

Add 1ul DpnI and incubate at 37C for two hours

Transformation

Add 1ul PCR product to 100ul competent cells. Add the remaining PCR product (24ul) to another eppendorf of 100ul competent cells

Follow standard transformation protocol

Restriction Digest

Incubate at 37oC for 60 mins - BamHI has star activity so it cannot be left for long

Component	Volume/µL	Component	Volume/µL	Component	Volume/µL
C#	5	C/D/H/M	5	E#/H#/P#	5
3.1 NEB 10x buffer	2	2.1 NEB 10x buffer	2	3.1 NEB 10x buffer	2
Bam HI	0.5	PstI	0.5	Bam HI	0.5
(no 2nd RE)	-	EcoRI-HF	0.5	EcoRI-HF	0.5
MilliQ	12.5	MilliQ	12	MilliQ	12

Gel electrophoresis of restriction digest

Gel Photo

Sent for sequencing:

Sequence	Concentration	Label Assigned
E* 1	45.1	SBDNaseDsbA1
J* 1	62.4	SBDspBDsbA1
C# 1	64.5	33LsrGFP1
H# 3	37.3	BMccS3
E# 4	67.3	BDNaseDspA4
P# 1	179.2	BDNase1
D* 3	56.7	SBLsrHolin3
H* 2	45.9	SBMccS2
M* 2	53.1	SBArtE2

Transformation of E. coli with ligation products from day12 (06/07 PCR and interlab sequences) using the standard protocol described by diagrams for day 3. Transformed E. coli then plated and incubated overnight

Day 14

Group A

C*, G*, H*, L*, N*, M* are biobricks

G*, J*, D* Sent for resequencing

A*, B*, F* I* K* - gradient PCR - currently in the freezer, run gel electrophoresis in afternoon, doing staining tomorrow

E*, J*, D* - redid PCR trying Q5 mastermix from 2014 and 2015 (gel poured for electrophoresis tomorrow)

Group B

C# - redo PCR (sequencing didn't not give expected resuts)

A#, B# - gradient (already currently in freezer - need to put onto gel)

C#,D# - colonies

Group C

E#, H#, P# - successful sequencing (with exception of point mutation in plasmid)

I# and F# - gradient PCR

M#, O# - redo from PCR

J#, K#, L#, N#, G#, P#, O# - colonies

G# - wait for G* to come back and QC if successful

(if QC doesn’t work again, treated with dpn1, gel, excise, clean and add buffer and ligase, transformation)

Is DH5alpha suitable for QC?

week, grow up competent cells for 2 E coli strains:

FliC deletion strain - FliC exported through Fla export system
MG1655 - close to WT E. coli - more tolerant (characterise in MG1655)

* primers are the templates for J#, I#, F#, G#

Results of sequencing:

Sequence	Result
E* 1	Forward read aligns, ask to repeat VR. Aligns to MccS (which is actually H*). Check the size of the PCR fragment. Redo PCR
J* 1	Forward and reverse reads align but highlight a point mutation at 847 but looking at the chromatogram there is also a peak for G.Send another, preempt another won’t work so start PCR
C# 1	Both reads are of insufficient quality. Look at the gel (sizes are off - two bands entirely diff size but both >2k - wrong)
H# 3	Forward read aligns but reverse read of insufficient quality. Potential deletion at T297 between promoter and RBS but otherwise should be fine. Point mutation in plasmid stock?
E# 4	Forward read aligns but reverse read of insufficient quality. Potential deletion at T297 between promoter and RBS but otherwise should be fine.
P# 1	Forward read aligns but reverse read of insufficient quality. Potential deletion at T297 between promoter and RBS but otherwise should be fine.
D* 3	Both reads align but there is a deletion at 2205. Send another.
H* 2	Both reads align.
M* 2	Both reads align.

G* has a 70bp insert at the start of the coding region (even though it’s been quikchanged). Send another.

PCR To Redo

E*, J*, D*

To Sequence

J* (not J1, J6 or J2) - J4

D* (not D3, D1 too low) - D2

G* (G4, G3, G2) - G4

To Overnight

The following tubes were put in incubator with selected colonies for over night culture (1x unless specified otherwise):

3x 3.1 C#	G#	3x P#	3.1 O#
3x pBAD33	C#	3x 3.1 D#	22A
pSB1C3	D#	3x 22K	20K
3x K#	L#	3x N#	3x J#

Day 15

Group A

Check sequencing data for G*, J*, D* :

G* = large anomalous insert at start of coding sequence → send another (G3) but also redo PCR verdict: G*3 sequencing data correct - no need further PCR
J*4 = biobrick
D* = large deletion in sequencing data → new batch currently being PCRed

(A,B,F,I,K)* - stain gel → visualize gel → 09/07 gradient PCR turned out to have no amplified products at all

(E,J,D)* - load dye → run gel → stain gel → visualize gel → only D* yielded bands → excise bands for (10/07) PCR products → cleanup → digest

Quikchange J*4 under gentle conditions to remove BspHI restriction site within its DspB sequence → wrong procedure was carried out (forgot to do DnpI enzyme incubation, and was PCRed despite quikchange products being meant to be directly transformed) → need to redo

pSB1C3 plasmids made from (09/07) overnight cultures → miniprep → digest with EcoRI, SpeI → gel poured and awaiting electrophoresis on Monday

E* was PCRed again using both standard and DMSO PCR protocol → but gel was frozen before excision and hence messed up → need to redo

J*4 QuikChange

Standard PCR protocol using QuikChange primers and following conditions:

Stage	Temperature	Time
Initial denaturation	98	30sec
Denaturnation	98	10sec
Annealing	65	30sec
Extension	72	3min
Final extension	72	2min

Group B

(A,B)# - stain gel → visualize gel → no bands for (09/07) gradient PCR products

D# - load dye → run gel → stain gel → visualize gel → excise bands for (10/07) PCR products → extract → digest → not cleaned up yet

(C,D)# - miniprep from (09/07) overnight culture → 10uL digested but not gelled yet

pBAD33 in (09/07) overnight culture - miniprep → digest → visualize → excise → fresh pBAD for ligation

Group C

(I, F)# - stain gel → visualize gel → no bands for (09/07) gradient PCR products

(M,O)# - load dye → run gel → stain gel → visualize gel → excise bands for (10/07) PCR products → extract → digest → not cleaned up yet

(J,K,L,N,G,P,O)# - miniprep from (09/07) overnight culture → 10uL digested but not gelled yet

More pBAD/HisB needs to be made → heat shock → plate colonies

Over the Weekend

Overnight cultures for plasmids setup

Quikchange for J*4 - parameter testing (previously we used the full 61.4ng/uL for quikchange - may be the reason why it never worked (for Q5MM’s efficacy, NEB recommends a total of 1-25ng of plasmid DNA in a 25µL reaction mixture)

1µL of 61.4ng/µL J*4 diluted into 60µL total volume to make 1.02ng/µL J*4

3 setups were made - 1, 5, 10µL of 1.02ng/µL J*4 at two annealing temperatures (65 and 70 degC) - making 6 tubes 3 minute extension time

Also E* and E*DMSO PCR

Week 4

Day 16

Leon

Minipreps for plasmids → concentrations noted on each tube

10µL of pSB1C3 digested
15.1µL of pBAD33
30µL of pBAD/HisB

Run E*, E*DMSO on the gel → bands very faint → both bands combined into one tube for gel extraction

Split into two tubes at the Binding Buffer step → at the end, one tube had 7ng/µL and the other had 9ng/uL of DNA

React J*4 QC with DpnI: all 24µL of all 6 tubes of J*4 from the QC PCR reaction placed into digest mixture (made up according to DpnI search using NEBCloner protocol desginer). Buffer = CutSmart; 2 hours incubation

Clean up digests done on Friday → cleaned up and ready for ligation

Digest plasmids and both tubes of E*

Day 1 (overnight) procedure carried out for the preparation of competent DH5alpha, MG1655, deltaFliC E. coli

Group A

Failed Gel

Helen and Drizzy: gradient PCR of A#, B#, F#, I#

A# used up the last of the 2014 Q5 MasterMix

Failed Gel

Mabel and Ria: normal PCR of A*, B*, F*, I*, K*

Mabel: prefix + post suffix holin primers, annealing temp = 58℃ (so set PCR to 56℃)

Ria: pre suffix holin + suffix primers, annealing temp = 63oC (set PCR to 61oC)

All failed - no DNA bands

Ria

Gel Photo

First gel of digests from Day 15 overran, re-do digest and gel electrophoresis etc.

Day 17

Plasmid Backbones

Dephosphorylation

1µL CIP added to heat inactivated pSB1C3, pBAD33, pBAD/HisB; incubation for 1 hour.

Enzymatic Reaction Cleanup

After cleanup, NanoDrop shows conc all are about 30ng/µL.

Ligation

All 29µL of each insert used, with 7µL of plasmid.

June

10/07 PCR Batch (D*, D#, M#, O#)

Ligation set up and left overnight.

13/07 PCR Batch (E, E DMSO)

Enzymatic reaction cleanup completed. Ligation set up and left overnight.

Preparation of Competent Cells

DH5alpha, MG1655, deltaFliC

Protocol

OD when incubation stopped:

DH5alpha - 0.302
MG1655 - 0.345 (achieved in one hour); 0.902 (1.5 hours - overshot because used more than 1mL, also wildtypes seem to grow faster than DH5alpha)
deltaFliC - 0.350; 0.805

The 0.902 MG1655 are labelled MG; the 0.345 MG1655 are labelled MGA and MGB.

The 0.805 deltaFliC are labelled with an extra dot • on the tubes.

Sequencing

Construct	Successful	Label assigned (1st sequence)
pBADArt175DsbA	K2	Art175DsbA2
pBADArt175Fla	N3	Art175Fla3
pBADArt175	O1	Art1751
pBADDNase	P2	DNase2

PCR of C#, D#, L#

Worked beautifully.

PCR of A, B, I, F, K*, A#, B#, F#, I#

K*: given to Chris for Phusion polymerase, will get results by the end of the week

Run gel of gBlock stock on 1% agarose with SYBR safe DNA Gel Stain in LAB buffer - no bands i.e. the working stock possibly has no DNA!

Nanodrop of gBlock stock: A = 7.9 ng/μl, C = 7.7 ng/μl (i.e. there is DNA)

N.B. A, B, I and F are our 4 largest gBlocks which we cannot amplify.

Driscoll's work: A, B, I and F attempted on a MgCl2 gradient of 0.5mM, 1mM, 2.5mM and 5mM. Ran PCR but was told that there was no DNA in the solutions so didn’t run gel, the PCR products currently sit in freezer but don’t think it’s worth it to run a gel as there is clearly nothing or not enough in the G-block stocks. The plan was to also to run a single PCR with A,B, F and I with 1% Triton X 100 and with double primer concentration but didn’t get around to doing it after hearing the results of the straight G block gel.

Helen: A# attempted with 10% DMSO. B# attempted with 10% glycerol. F# attempted with 5% formamide. I# attempted with 10% formamide. Ran a gel of these PCRs, and none showed any bands.

Day 18

Check Plates

As expected, MGA and MGB are more competent (grew more extensively on antibiotic plate) than MG(OD=0.9 when taken out of flask)

FliC0.35 also more competent than FliC0.8

Transform Ligated Products into DH5alpha

E*, E*DMSO, D*, D#, M#, O#

Ligation

Set up ligations for C#, D#, L# (PCR batch 14/07)

Mixed up the samples so will be ligating tomorrow.

Overnight Culturing

QC J*4 (3) grew colonies.

Conditions used for its Quikchange PCR was: 10.2ng plasmid DNA in the 25µL PCR reaction.

By the end of the day, a few more of the J*4s also had colonies

J*4 (3) and J*4 (1), which seemed to had the densest colony growth, had overnight cultures set up (3x each) in Chl LB broth.

PCRs

Same as previous but re-diluted primer stock and used 1µl of IDT gBlock (i.e. 10ng of DNA) for A*

Run at 2 different cycles: either 98 and 72degC for 2 mins or 98, 72 annealing, 65 for 1 min and 72 for 1 min for extension.

Gel: SYBR safe and agarose in LAB - see 14/07.

NO BANDS, but the primer dimer band for the 65 degC extension was brighter when visualised with blue light.

Leon to email IDT about further recommendations.

Transform completed plasmids into FliC and MG1655 Competent Cells

N#, P#, H# and E#

Making up M9 modified media

We made it up using the recipe from this website for 500ml. We filter sterilised all components except milliQ water and stored it on the bench with LB.

Day 19

Mini-prep overnighted J*

6 colonies grown overnight

Alterations to protocol:

Spin down entire volume of LB broth to ensure all E. coli are used in the extraction.
Pulse after spinning down all the E. coli to remove excess LB
Heat elution buffer to 55 degC before eluting 50ul, spin down, re-pipette filtrate into HiBind column and spin down again

Nanodrop values (values are quite consistent - good idea to apply all modifications for any mini-preps in the future)

Eppendorfs in freezer drawer awaiting potential sequencing

note: (1), (2), (3) are from plate 1 (corresponding to QC PCR at 70degC annealing temp, 1ng plasmids); (4), (5), (6) are from plate 3 (corresponding to QC PCR at 70degC annealing temp, 10ng plasmids)

	[DNA] (ng/µl)
J*4 (1)	175.2
J*4 (2)	172.2
J*4 (3)	153.3
J*4 (4)	194.5
J*4 (5)	181.0
J*4 (6)	173.9

Diagnostic restriction digest of J* + gel electrophoresis

Gel Photo

20µl reaction for 90 minutes in PCR tubes

See protocol

Gel: 1% agarose, 120V

Ladder	J*1	J*2	J*3	J*4	J*5	J*6

Lack of ladder but all consistent - send for sequencing tomorrow

Pick Colonies

Pick colonies of N#, P#, H# and E# (delta FliC and MG1655) and culture overnight

Pick colonies for E*, E*DMSO, D*, D#, M#, O# and culture overnight

PCR

O# and QC G*

QC PCR protocol

Ran PCR on 1ng, 5ng, and 10ng of template DNA with 3-minute extension time.

Digest PCR products in DpnI, according to the protocol here

^has been done. Gel currently running.

Transform all the samples into separate cultures of competent DH5alpha

^has not been done

Re-do C#, D#, L# PCR

Restriction Digestion and Ligation

Restriction digest done on R and L.

Set up ligations for C#, D#, L# (PCR batch 14/07), running over night. After the gel only two managed to be extracted, one of C# or D# (R#) and L#.

Day 20

Leon

3 frozen samples each were stored in the -80 degC freezer for E# (FliC), E# (MG1655), H# (FliC), H# (MG1655), N# (FliC), N# (MG1655), P# (FliC), P# (MG1655) following the overnight cultures that were set up on 16/07

They are in our usual freezer drawer, and within it the bottom most slot of the third column from the front, in a box labelled “iGEM transformed cells, MG1655 and FliC”

Raffy

Sent J*4 (4) for sequencing

Mabel and Duke

Miniprep - E*, E*DMSO, D*, D#, M#, O#

Miniprepped overnight cultures from June and Raffy above. Plasmids were eluted with 50ul of elution buffer.

Nanodrop:

Plasmid type	ng/µl
D#	72.2
D# 2015 e1	160.6
D# 2015 e2	42.0
D# 2015	65.6
D* 2015	238.4
D* 2015 e	40.4
D* 2	288.9
D# e	31.2
D* 2e	378.7
E*1	333.9
E*1 e	287.5
E*2	370.6
E*2 e	367.1
M#e	50.5
M# 2015	50.8
M#e 2015	62.0
O#	49.9
O# 2015	82.7
O# e	44.1

Restriction digest of minipreps completed - find the protocol here 20µl reactions.

Ria and Lychee

Transform R#, L# into DH5alpha. R# is Chl and L# is Amp antibiotic

Helen and Kyle

Digested plasmids for successful PCRs, plasmids not yet cleaned up.

Over the Weekend

Agar plates from 17/07 transferred to cold room

Sequencing data for J*4(4) received (order no: 4254194) - QC successfully corrected desired base, but also overzealously introduced two repeated sequences corresponding to the primer sequence 1. after the QC area of interest and 2. at the start of the DspB sequence (see SnapGene file: QC J*4 wrong). Colonies on plate 3 should all have wrongly QC-ed plasmids as a result.

Will send either samples (1), (2), or (3) for sequencing (as they are from plate 1 - PCRed under different conditions), and also overnight culture some colonies from plates 4, 5, and 6 as they were QC PCRed with a lower annealing temperature (lower possibility of wrong extension?)

Overnight cultures set up:

Number of Tubes	Plasmid of Interest
3	L#“
3	“R#”
3	F*
3	I*
4	QC J*4: (A), (B) are from plate 5e (PCRed at 5ng plasmid, 65degC annealing) (C), (D) are from plate 6e (PCRed at 10ng plasmid, 65degC annealing)
1	Interlab 22A
1	Interlab 22K
1	Interlab 20K

On 20/07 Day 21

Interlab cultures - transfer 700uL of the respective cultures into appropriately-labelled microcentrifuge tubes and add 300uL of 60% glycerol to each of them. Vortex for even mixing and flash freeze at -80degC. The rest of the cultures can be miniprepped to replenish plasmid supplies for further transformation into MG1655 and deltaFliC.

All other cultures.

Week 5

Day 21

Post-digest cleanup → ligation

For C#, D#, L# from the 16/07 PCR batch

Sequencing of QC J*4

J*4 (4) sequencing data shown to have many extra inserts, despite the desired mutagenetic site actually being correctly replaced. Two paths will be pursued:

J*4(1) (which is from plate 1 - another PCR condition (1ng plasmid)) to be sent for sequencing - obtain 2.5µL plasmids and dilute 1:1 in 2.5µL of Milli-Q. Sent for sequencing using VF2 and VR primers.
J*4(1) (which is from plate 1 - another PCR condition (1ng plasmid)) to be sent for sequencing - obtain 2.5uL plasmids and dilute 1:1 in 2.5uL of Milli-Q. Sent for sequencing using VF2 and VR primers.

If and when the sequences are correct, we will save the corresponding tube as a complete BioBrick (annotating that it has been QuikChanged), digest out the insert, cleanup, PCR the NcoI restriction site in, digest, and finally ligate into pBAD/HisB to make a functional plasmid.

Sequencing Data Received

K#1 confirmed - refer order number 4253587

Gel electrophoresis confirmation of 17/07 digested miniprep products

Plasmids -

pSB1C3: D*, E*
pBAD33: D#
pBAD/HisB: M#, O#

After which, send for sequencing depending on degree of success. Unfortunately the gel ran too far and ‘ladder’ didn’t show up, which suggests that it is actually a mislabeled tube of loading dye. Re-made digests as only 3 ul of original is used. Will run gel again tomorrow with fresh ladder.

Miniprepping of 19/07 overnight cultures

Miniprep L#, “R#”, F*, I*, QC J*4

For the rest - miniprep the full volume (see “upgraded” protocol: spin down cells → decant → resuspend in smaller volume before beginning the miniprep; pre-warm elution buffer to 55degC; run the same elution mixture through the column twice for maximum extraction)

Digest 10µL → gel run (gel is now in cold room, awaiting staining and visualization tomorrow)

Transformations

K#1 → MG1655, deltaFliC

Plate Streaking

Frozen stocks of MG1655, deltaFliC N#, P#, H#, E# streaked on agar plates.

Day 22

Sequencing Data Received

QuikChange J*4 (1) and ( C ) returned correct sequences (refer to order nos 4254450 and 4254692 respectively). Both are now stored in “Complete BioBricks” box.

PCR Amplification to make J# from QuikChanged J*4[pSB1C3]

J*4(1) was used; 3 min extension time (lol too long)

Transformations

Transform C#, D#, L#, O# into DH5alpha

Transform pBAD33, pBAD/HisB into MG1655, deltaFliC for positive control.

Overnight Cultures

From newly transformed plated cultures:

MG and FliC - InterLab, K#
DH5alpha - (+) and (-) controls for InterLab

From streaked cultures:

MG and FliC - E#, H#, N#, P#

Run Gel for miniprepped JQC, F, I*, R#, L#

Gel Photo

F*, I* insert band very very faint - most probably because vector:insert ratio is about hundredfold

→ redo direct digest using 2µL (20ng) stock gBlock, ligation with less plasmid (~10ng)

Kyle already doing C#, D#, L#

Top: J*4A, J*4B, J*4C, J*4D, F*1, F*2, F*3, I*1, I*2, I*3, L#1, L#2, L#3

Bottom: R#2, R#3

Sent F*1, I*3 for sequencing to find out what is going on with confusing 3kb/2kb double band. Results (order no: 4254965) shows both samples containing only blank pSB1C3.

Restriction digest and ligation of A, B, F, I, and K* directly from the G blocks

Digested and dephosphorylated psb1c3 with EcoRI HF and PstI HF, made 1ng/µl psb1c3 solution.

Digested A*, B*, F*, I*, and K* with EcoRI HF and PstI HF, made 15ng/ul insert solution.

Cleaned up both inserts and vectors.

Set up ligations with 15ng insert and 5ng vector.

Duke and Lychee

Gel Photo

Managed to successfully run the gel for the test digests failed on 20/07/15. Image of gel shows that digestion following miniprep failed, but the presence of 3kb bands suggests that the ligation was in fact successful. Could send them for sequencing to find out.

Strong 2kb and 3kb bands for D* and E

D# only showed 3kb band

M# and O# show bands slightly larger than 3kb

Gel was loaded in the following order:

1st row: -Ladder-D*IIe-D*2015-D#e-D*2015e-D#2015e2-O#e-D*II-E*1e-M#2015-O#2015-M#2015-E*2-D#-D#2015-
2nd row: -Ladder-E*2e-O#-E1*-D#2015e1-M#e

PCR of QuikChanged J*4(1) with primers for pBAD restriction sites

Gel Photp

Gel extraction EZNA carried out to combine both bands into one sample.

Day 23

Frozen Stock Preparation

K# in MG1655 and deltaFliC made into frozen stocks

Tasks Completed

Transformation of A*, B*, F*, I*, and K*

Digestion of J# and pBADHisB, then ligated.

Overnight cultures of C#, D#, L#, O#

PCR amplification of O#

Test Digest for DIIe, E1 compared with digest of completed BioBrick N*3

Test digest shows no problem with restriction enzymes, as completed BioBrick was successfully digested.

Recommendation - for the parts that are going to be redone, it is recommended that one person takes it through.

Day 24

Overnights

Pick colonies and overnights for A*, B*, F*, G*, I*, and K*

Set up overnights for toxicity assay again: E#, H#, K#, N#, P#

Transformations

Transformation of empty pBAD33 and pBADHisB into FliC and MG1655

Transform QCJ# into DH5alpha - Colonies grown

Sequencing Data

M#e and D#2015 sequencing data shows no insert (from 21/07 miniprep digest gel run photo)

Miniprep C#, D#, L#

Test-digest → gel run:

Ligations

Ligate O# into pBAD/HisB

Done overnight, ready for day 25 transformation.

Day 25

Miniprep

A*, B*, F*, I*, K*, G*QC

Sample	ng/µl
A*1	56.8
A*2	65.7
A*3	57.5
Be*1	56.6
Be*2	101.7
Be*3	52.0
Ge*1	30.2
Ge*2	55.2
F*1	362.5
F*2	111.1
F*3	182.5
I*1	335.6
I*2	411.1
I*3	414.1
K*1	373.9
K*13/td>
K*3

Gel Electrophoresis of minipreps

Ladder	A* 1-3	B* 1-3	F* 1-3	G* 1-3	I* 2	I* 3	I* 1	K* 1-3

Gel Photo

G* and F*2 to be sent for sequencing on Monday.

Transform

J#QC → DH5alpha - Amp

DsbADNase (from iGEMHQ) → DH5alpha - Chl

Over the Weekend

Streak plates

EHKNP# - 2 species (10 plates) Amp

(+)(-) - 3 species (6 plates) Chl

Overnight Cultures

pBAD/HisB in MG1655, deltaFliC; pBAD33 in MG1655, deltaFliC, O#

Miniprep

Miniprep of QCJ#

Week 6

Day 26

To Do

Make up more modified M9 media
Melt agar and agarose
Send G*1, F*2 for sequencing
Miniprep + digest O#

Digest J# QC, run gel of O# and J# digest

Gel Photo

No insert for O#, Sent J#3 for sequencing

Day 27

Raffy

PCR C,D,L,M,N#

Day 28

Gel run for 28/07 PCR products, Band Excision, Clean Up

C#, D#, L#, M#, O#

Digest + clean up

pBAD33, C# and D# with BamHI

pBAD HisB, L#, M# and O# with NcoI and PstI

	ng/µl		ng/µl
C#	1.1	O#	1.3
D#	0.4	pBAD 33	6.1
L#	0.3	pBAD HisB	3.9
M#	1.1

Ligatation

C# and D# with pBAD 33

L#, M# and O# with pBAD HisB

See protocol here.

	Volume/µL
Insert	34
T4 DNA Ligase	1
DNA Ligase Buffer	10

	C#	D#	L#	M#	O#
Plasmid	2.5	1.0	0.1	0.2	0.2
Milli-Q	2.5	4.0	4.9	4.8	4.8

N.B. plasmid:insert ratio is significantly lower than 1:3 - will see if this leads to better ligation results

Success

QC J#3 sequence success (ref: 4256167)

Transform successful QC J#3 into MG1655, deltaFliC

Day 29

Sequencing Data

G*e2 QC sequence success (ref: 4256185)

→ PCR it to make #

Gel band excised and stored at -20degC

Transformations

Transform expression contructs ligated yesterday into DH5alpha

C#, D#, L#, M#, O#

(only 1µL plasmid used, may potentially not work)

Transform QCJ# into MG1655, deltaFliC.

Plate out BioBrick (T4 Holin) agar stab

Plated on Amp (pSB1A2)

Day 30

Overnight cultures

For QCJ# MG and deltaFliC; C#; T4 Holin BioBrick

Transformation into DH5α

C#, D#, L#, M#, O#

To Do

Cleanup; Digest; Ligate QCG#

Over the Weekend

Make stocks of J#QC

Innoculate C# D# L# M# O#

transform #s and interlab for characterisation

Make stocks of J#QC

Overnights of #s and interlab for characterisation (all LB media)

Miniprep C# D# L# M# O# and Bba

Week 7

Day 31

Miniprep

Gel Photo

Minipreps for C#, D#, L#, M#, O# → Test digest and run gel (done yesterday)

Send D#1, L#3, O#1 for sequencing

Day 32

Sequencing Results (4257633):

pBADHisB Art-175 (O#1) - success
pBAD33 Lsr Holin (D#1) - 1 mutation, ask them to resequence
pBADHisB YebF Art-175 (L#3) - 1 mutation, ask them to resequence

Day 33

George

Miniprep G#

Helen

PCR K # → * KHTS primers, annealing temperature 72*C, extension time 2 mins

Cleanup C#, M# digested → ligate

Mabel and James

Set up overnights - add J# to them

All in LB Chl or LB Amp (inc. interlab)

Day 34

Kyle

Sequences confirmed (4257633) for D#, L#, O#. Therefore transform into MG, FliC.

Transform C* into MG, FliC

Helen

Transform ligated C#, M# into DH5alpha.

Day 35

To Do

i)

If colonies have formed from the transformation of ligated C# and M# in DH5alpha, pick colonies and set up overnight cultures in the afternoon. Someone will need to come in on Saturday to miniprep these cultures, consult Mabel for tips and tricks on miniprepping if necessary.

James and Lychee performed minipreps on Saturday and will update on where the miniprepped DNA are in the freezer.

ii)

If colonies have formed for the MG1655 and deltaFliC with C*, D#, L#, O#, pick colonies and set up overnight cultures in the afternoon. These cultures do not need to be miniprepped. The next day, make two tubes of frozen stocks out of each culture, and also since it is now the end of the week, streak some of the liquid cultures onto agar plates supplemented with the appropriate antibiotics. If unsure about where frozen stocks of expression cultures should go, consult Mabel.

James and Lychee made frozen stocks out of these cultures on Saturday.

iii)

E* has yet to be inserted into pSB1C3: PCR from gBlock solution.

E* PCR failed. We shall consider PCR-ing directly from gBlocks again, or design long primers to PCR E* out of the completed E# (inserted in pBAD/HisB) plasmid.

iv)

We currently have D# (Lsr Holin), but not an endolysin needed to complete the Holin-Endolysin lysis system. In case our Art-E part (M#) again fails to successfully be cloned, procure BBa_K112806 [pSB1C3] from the 2015 Distribution Kit (Plate 3, Well 12K) and transform into DH5alpha. Subsequently we can potentially insert a promoter in front of it to complete the lysis system.

Colonies formed and the plate was transferred to the cold room.

v)

Since B* failed to PCR to give the Synbiota LasR and pLas parts that we needed, we will instead obtain them from the Distribution Kit as well. They are both in pSB1C3, LasR being BBa_C0179 in Plate 3 Well 6M, and pLas being BBa_R0079 in Plate 3 Well 5C respectively.

Colonies formed and the plates were transferred to the cold room.

vi)

G# miniprep yesterday showed no insert: Using QC G* as template, PCR using primers DspB forward and Post Suffix Holin to make it into G# for cloning into expression vector.

PCR succeeded, excised gel bands kept in freezer.

vii)

Continue with KHTS - ligate overnight, provided someone can put them in the freezer tomorrow morning.

Transferred into freezer.

viii)

Transform the part 2015 Kit Plate 2 Well 17M and then grow in agar plate. Silas will need to film doing the transformation and the plating process.

Completed.

Week 8

Day 36

To Do

Test digest C#, M#, if working send them for sequencing.
PCR E* (normally).
Cleanup G# from excised gel, digest (remember to digest some pBAD/HisB plasmid as well), heat inactivate, dephosphorylate the plasmid only, ligate the both of them.
Transform K* (KHTS).
Synbiota PCR

N.B. Our original gBlock solutions are 10ng/µL - use 2µL of that for the PCR

Day 37

PCR C# from C*pSB1C3, M# from M*
Why won’t E* PCR??
G# ligated into HisB (the tube was labelled G* into HisB)
K* transformed - check if colonies formed

Day 38

Out of Q5, so use Pfu - this involves adding nucleotides, different annealing temperatures - ask Chris or George

BioBricks

F and I with new restriction enzyme combination (XbaI, PstI-HF) - up to ligation overnight (Mabel)
PCR K# to K* (Kyle)

Expression

E# - try one more time but then if it doesn’t work come to Leon or Raffy to design long primers (Kyle)
G# pick colonies (Kyle)
C# and M# repeat PCR (Helen)

Day 39

Design (E,D)# to (E,D)* primers
Transform SBArtE2 into DH5alpha i.e. M* hopefully
Transform the ligated F*, I*s which were directly digested from gBlock using X, P
Miniprep G#

NB: did phusion go back in the freezer from which it came? Ensure it doesn’t go in our freezer.

Day 40

Pick M*, F* and I* colonies
PCR G* to G# and E# to E*
K* and C# ligated overnight - transform

Over the Weekend

Miniprep of F*, I*, K*

Nanodrop:

	ng/µl		ng/µl		ng/µl
F*1	353.5	I*1	464.1	M*1	204.2
F*2	430.0	I*2	428.4	M*2	148.3
F*3	351.3	I*3	363.8	M*3	189.1

N.B. I*1, I*2, M*1 and M*3 are not pure samples (curves on nanodrop had weird peaks) - these are the eppendorfs without smiley faces drawn next to the [DNA] on the side of the tube.

N.B.B. Mabel has a new PB for [DNA] on the QIAGEN miniprep kit ;)

Restriction digest: 20ul reaction, 37degC for 90 mins

M* also digested with XbaI and PstI-HF - assume that the insert would have the same MCS as F* and I* if they’re all meant to be for biobricks. See protocol here.

Gel: 1% agarose

Only needed ~35 mins to stain well - due to high [DNA]??

M*2 can be sent for sequencing (only ‘normal’ curve on nanodrop)

F*1	F*2	F*3	I*1	I*2	I*3	M*1	M*2	M*3

Gel Photo

Streak all frozen stocks - in two locations

Bottom most box in the 3rd column has a portion of the frozen stocks but not the new ones

New ones are in the topmost box in the second column

Overnights:

All interlabs (unnecessary duplicates of deltaFliC but nevermind) and extra 20K MG and 20K DH5
E# and J# + controls (duplicated)
P. putida

Week 9

Day 41

Last few bits of cloning

Transform C* (in complete biobricks box) into M51655 and RP437 (oops C* is already transformed and D* doesn’t exist)

Send M*, F* and I* for sequencing

→ F1, M2 and I3 sent

Day 42

PCR M# from M*
Synbiota PCR
O* (Art-175 on its own) isn’t a BioBrick - let’s make it!

Day 43

Test digest of C# -> run on gel -> send C#1dF for sequencing -> not right.

All C# miniprep values were very very low, probably due to tranformation into wrong strain. PCR C# tomorrow.

PCR G# -> run on gel -> gel extraction -> restriction digest.

Day 44

Carry on from gel extraction of G# - restriction digest -> clean up -> nanodrop (Kyle has values) -> ligation.

PCR C* to C# again.

Day 45

C# gel - excise bands, continue

G# ligation - This is in the front of our top freezer drawer. Transform this into DH5a

Week 10

Day 46

Constructs PCR

New primers arrived - but mistake made with M (thought it was # to * but actually need * to #, so using old primers ordered previously)

Construct	Fwd primer	Rev primer	Annealing temp.
P (# to *)	DNase HTS	Art E suffix	72
K (# to *)	DsbA HTS	Art E suffix	72
E (# to *)	DsbA HTS	Art E suffix	72
C (# to *)	Lsr Holin HTS F	Lsr Holin HTS R	71
M (* to #)	SMAP 29	Art E suffix	70

Also used Phusion Polymerase but primers are at 10µM instead of 25µM.

Ladder	T4 Holin	pLsr	C*	E*	K*	P*

Gel Photo

G# colonies picked, in overnight broth in 37 degree incubator. Plates are in the cold room.

Day 47

Molecular cloning of constructs

Gel extraction of C*, E*, K*, P* - eluted with 35ul of EB

Restriction digest of C*, E*, K*, P* and pSB-1C3 plasmid with XbaI and PstI-HF in a 50ul reaction, 37 degC for 1 hour at 300rpm

95degC heat inactivation for 30mins

Add 1µl CIP for pSB-1C3 only at 37degC for 30 mins

Cleanup - elute insert with 35µl EB buffer, plasmid with 50µl

Ligation set ups: 50ul reaction, use all 34ul of eluted DNA

Incubate at 16 degC overnight

N.B. There is some reaction mixture of M* to M# left over from 24/08 so will use this to run a gel with the synbiota PCRs.

Day 48

Kyle

G# miniprep -> nanodrop -> test digest

Mabel

M# mentioned in synbiota section

Transformation of C*, E*, K* and P* into DH5α

Plated transformations onto Chl plates - watch out for potential mixup of P* and K* in the low concentration bacteria plates

PCR of D# to D* using Pre prefix holin and post suffix holin primers, standard Phusion set up, annealing temperature 71degC

Day 49

PCR of O# to *, using new primers that arrived on 24/08 (thought they were for M# - * but were actually for O)

Forward primer: Art-175 HTS, Reverse primer: Art-E suffix

Diluted to 25uM, annealing temperature 71degC

Three different setups:

Phusion RDP protocol
Phusion RDP protocol with GC buffer from Chris (optimised for primers with a high GC content e.g. Rhodobacter genome)
Phusion RDP protocol with 5% DMSO

Template was not digested before PCR

RDP	RDP	GC buffer	GC buffer	DMSO	DMSO

Gel made with water…………………………………!@%*#%$^

E*, K* and P* (we actually already have C*) - 2 colonies picked from each plate, so total of 4 overnights per construct (high and low conc plates)

Day 50

Repeat PCR of O# to *, Phusion set up as on 27/08 but no DMSO or buffer change.

Miniprep of E*, K* and P*: all eluted in 50ul EB buffer

Nanodrop (adjusting volume added to digest to keep [DNA] roughly the same)

37 degC for 90 mins

Gel: 1% TBE agarose, 120V

it didn’t melt! YAAAAAAAAAASSSSSSS >8)

E*1	E*2	E* [ ] 1	E* [ ] 2	K*1	K*2	K* [ ] 1	K* [ ] 2
P*1	P*2	P* [ ] 1	P* [ ] 2	O#-* 1	O#-* 2	O#-* 3	O#-* 4

Excised all O* bands (now in freezer)

K* [ ] 1 can be sent for sequencing (insert length should be 1030bp)

Week 11

Day 51

Molecular cloning day YAYZ

Gel extraction of D* and O* PCRs

Digest of D*, O* (50ul set ups) and pSB-1C3 (100ul set ups) with XbaI and PstI-HF

Heat inactivate inserts and plasmid at 95degC for 30 mins

Add CIP to pSB-1C3 and incubate at 37degC for 30 mins

Clean up: inserts eluted in 35µl, plasmid eluted in 50µl

Ligations: 50ul set ups

All with 5ul of 10x T4 DNA ligase buffer and 1ul T4 DNA ligase

From the nanodrop values omitted O*3 because [DNA] is too low.

Incubate at 16degC for 16 hours

PCR G* to G#

3 different conditions: RDP protocol, RDP protocol with GC buffer, RDP protocol with 5% DMSO

Gel: 1% TBE agarose, 80V

5% DMSO	5% DMSO	GC buffer	GC buffer	RDP	RDP

Something is wrong with the camera :/ - everything except the DMSO PCRs have been excised

Transformation of pSB-1C3 into DH5α - plate onto Chl plates

Accidentally transformed all the DH5α - need to make DH5α competent cells from plate in cold room (given on 23/07 so not sure how successful this is going to be…)

To Do

Made 5ml of Chl antibiotic stock (into EtOH)

Overnights: re-pick from E* and P* plates into Chl LB

3 colonies per plate, 12 overnights in total

Day 52

Mabel

Miniprep E* and P* overnights - all eluted in 50µl buffer

20µl digest with XbaI and PstI-HF

Gel: 1% TBE agarose, 120V

1kb+ ladder	P* 1-3	P*e 1-3	E* 1-3	E*e 1-3

June

Making DH5alpha Competent Cells

20 eppendorf tubes are restocked in -80C

Transforming O* and C*

Plates are : O*1, O*1e, O*2, O*4, D*1, D*2, D*1e, D*2e
x10 conc plates gave good colonies

Picking colonies and setting up overnight cultures for psb1c3 transformed in DH5alpha

Miniprepping E* and P* overnights

Day 53

Repeat digest of E* and P* cos I’m an idiot and i forgot to write down what they were

E*2	E*3	E*e 2	P*2	P*3	P*e 2

Send E*2 and E*e2 for sequencing forward read only - whichever one comes back will get a reverse read.

Miniprep of pSB-1C3 - all eluted in 50µl

Digest with just XbaI to linearise for gel

1% TBE agarose, 120V

pSB-1C3 1	pSB-1C3 2	pSB-1C3 3	pSB-1C3 4	pSB-1C3 5

Mystery of the massive bands: case closed.

The massive band is linearised pSB-1C3!!

Day 54

Miniprep of pBAD HisB, D* and O* overnights - all eluted in 50µl EB

Digest: * with XbaI and PstI-HF in cutsmart buffer, HisB with NcoI in 3.1 buffer

Moved into freezer - run gel tomorrow

Moved E*2 into biobricks box :)

Day 55

Digest of M#, G#, pBad HisB

G#: BspHI, PstI-HF in 3.1 (should be cutsmart but Mabel forgot to write the buffer for that set of enzymes)

M#: NcoI, PstI in 3.1

pBAD HisB: Nco, PstI in 3.1

Digests from Kyle

Heat inactivate at 95 degC for 30 mins

Add CIP to pBAD HisB for 30 mins at 37 degC

Clean up: HisB eluted in 50µl, inserts in 35µl

Ligation into pBAD HisB: all with 5µl buffer and 1µl DNA ligase

N.B. The G# ligations are slightly over 50ul - diluted HisB too far in digest

Leon to move ligations onto Mabel’s orange rack in the -20 freezer.

Molecular cloning

Run gel (1% TBE agarose, 120V) of O*, D*, pBAD HisB and remaining P# - * PCR from 24/08 with 1kb+ ladder

D*1	D*2	O*1	O*2	pBAD HisB	P# - *	P# - *

P# - * PCR has been excised and gel extracted - on orange rack in freezer

Send O*1 (as Art-175 1) and K* high conc.1 (as Art-175 DsbA 1) for sequencing with correct primers

Characterization

Plan

Overview of sequences in pBAD

pBAD 33 LasR Holin	A#	No PCR
pBAD 33 LasR sfGFP	B#
pBAD 33 Lsr sfGFP	C#	Overnight Culture
pBAD 33 Lsr Holin	D#	Miniprepped, awaiting gel --> sequencing
pBAD HisB DspB YebF	E#	Transformed into MG1655, deltaFliC
pBAD HisB DspB	F#
pBAD HisB MccS	G#	QC in progress
pBAD HisB DspB Fla	H#	Transformed into MG1655, deltaFliC
pBAD HisB DspB DsbA	I#
pBAD HisB Art-175 DsbA	J#	QC introduced extra sequences (order no 4254194); send another
pBAD HisB Art-175 YebF	K#	Sequence confirmed (order no 4253587), awaiting transform
pBAD HisB Art-E	L#	Overnight culture
pBAD HisB Art-175 Fla	M#	Miniprepped, awaiting gel --> sequencing
pBAD HisB Art-175	N#	Transformed into MG1655, deltaFliC
pBAD HisB Art-175	O#	Miniprepped, awaiting gel --> sequencing
pBAD HisB DNase	P#	Transformed into MG1655, deltaFliC

Currently completed pBADs (frozen stocks of plasmids in MG1655 and deltaFliC made):

With secretion tag - E# (DsbA DNase), N# (Fla Art-175)

Toxicity assay: dilute down with varying conc of inducer

Different growth conditions - flagellar need TB media 30degC

Fluorescent

Plate streaking → colony picking → liquid culture → spin down → supernatant contains proteins

Protein purification using His-tag affinity (SDS-PAGE then Ni(II)/Co(II) column/nickel-chelated horseradish peroxidase? Or use Western Blot using antibodies selective for His-tag?)

Nanodrop at 200nm and 280nm to measure protein concentration in eluent

Assaying for DNase:

Culturing on DNase test agar (if secretion tag works, no need to purify protein) → visualize DNA hydrolysis either by i) HCl flooding to precipitate DNA out ii) using a dye such as toluidine blue
If we want to make use of the purified proteins - need to have a solution-based assay that can detect polymerized DNA vs hydrolysed DNA?

Assaying for antipseudomonas activity: the Chang paper used PAO1 as target (for DNAse, MccS):

Grow PAO1 in 150uL medium in 96-well, incubate 24h with peg lids → rinse pegs with PBS → immerse pegs in plate containing purified DNase → crystal violet staining (alternatively maybe can just grow biofilms at bottom of wells - harder to wash)
Grow PAO1 similarly, then immerse in culture of DNase-secreting E. coli

Crystal violet staining: stain, rinse, de-stain, measure absorbance at 575nm

Assaying for Art-175: basically incubate PA (the original Art-175 paper used PA14) with purified Art-175 (or supernatant since our Art-175 has secretion tag) and monitor OD at 600nm over time (refer to paper)

Without secretion tag - H# (MccS), P# (DNase)

Need to lyse cells (buffer or sono?)

MccS - Chang’s original paper simply tested for antibacterial activity by adding purified MccS to a culture of PAO1

Secretion tag yes/no study for DNase since we have both
See if DNAse present extracellularly even when there’s no secretion tag

Cell Growth

Week 5

Day 23

Toxicity Assay

Arabinose induction of pBAD expression was studied at various arabinose concentrations to test for toxicity of proteins encoded by our inserts.

Each well has total volume of 200µL - culture (1/200 dilution), arabinose, media.

96-well plate loaded in triplicate according to the following plan:

DF = deltaFliC

MG = MG1655

Percentages denote final w/v concentration of arabinose in each well

Initial monitoring shows that the wells containing only Blank LB has increasing OD600 over time - likely to be contaminated LB, makes the rest of the dataset unreliable as well as the cells were inoculated using the same bottle of LB.

Forgot to turn on incubator so the entire curve was done at room temperature.

Day 24

Toxicity Assay

Forgot to turn on incubator so the entire curve was done at room temperature.

Day 25

Toxicity Assay

Wrong antibiotic in supposed positive control wells.

Temperature - 37degC.

Week 6

Day 26

Toxicity Assay

96 well plate layout

Pipetting triplicates only - 1 eppendorf split between 3 wells

Eppendorf Set Up	Volume (µL)
LB + Amp OR Tryptone + Amp (both 1:1000 dilution of Amp)	985
20% OR 2% OR 0.2% Arabinose	10
Stationary phase overnight culture	5

Plate reader: toxicity assay script

90 cycles, 900 seconds
200rpm shake when idle
LB Amp: 37degC
Tryptone Amp: room temp (forgot to set to 30

Day 27

Reapeat of Yesterday

Toxicity assay, in Tryptone Broth at 30degC

Day 29

Toxicity Assay

Replace 0.002% Arabinose with MilliQ.

85 cycles, 900 seconds, 30degC

Week 8

Day 36

Toxicity Assay

It has been previously established for E#, J#, K#, N#, P# that 0.2% arabinose incubation is non-toxic (for H# deltaFliC, toxic even under 0.002%). For all constructs, 0.3% and 0.4% are both quite toxic levels of induction.

We now want to investigate whether 0.2% arabinose induction is toxic for new constructs D#, L#, and O#.

Day 37

Set up 1%, 2% and 4% arabinose toxicity assay for (E, H, J, K, N, P)# and two more rows for controls.

Protein Purification

Week 6

Day 29

TCA Protein Precipitation

For the protocol see here.

Using old supernatants, and potentially spin down the cells from yesterday.

Couldn’t have worked because arabinose wasn’t added to cell cultures → suspension wouldn’t have had secreted proteins.

Day 30

Secretion Assay

Culture cells to mid-log in LB and TB

For LB, keep in 37degC throughout

For TB, once reaching mid-log, move to 30degC

Culture for another 1 hour

Take 1.6mL of the culture, centrifuge, keep supernatant and discard cells

TCA precipitation protocol

(can refer to Andi’s by going back out to this week’s folder)

Catch Jia when she’s around and get her to show us where the SDS-PAGE stuff are.

Week 7

Day 32

Secretion Assay

EJNK# in both strains, 1.5 hours incubation at LB at 37degC (1:10 dilution of overnight stationary culture), then 4 hours incubation with 0.2% final conc of arabinose.

Day 34

TCA-Precipitate

TCA-precipitate cell-free supernatant of EJKN# and run SDS-PAGE

E#MG, N#MG no secretion; J#MG, K#MG, J#DF, K#DF, N#DF lysed; E#DF successful secretion.

Week 8

Day 36

Supernatant Acquisition

Wash 1.5mL of overnight cultures of E#, J#, K#, L#, N#, ctrl MG1655 in antibiotic-free LB Broth twice and grow them in 1:50 dilution with 0.2% arabinose in conical flasks. Obtain 4x1.4mL aliquots of their supernatants after 4 hours incubation at 37degC.

Do the same for H# MG1655, except don’t obtain aliquots of its supernatant. Instead, obtain aliquots of culture so that we eventually can lyse the cells to obtain MccS from the lysate.

SDS-Page

Obtain supernatants of deltaFliC constructs from cold room - carry out standard SDS-PAGE protocol.

Day 38

Assaying

Run PAGE on the supernatants obtained from the M9 MG1655 cultures from yesterday. Do this first thing in the morning and hopefully as we move onto doing the interlab stuff the gel will develop enough to show some bands.

If bands formed on the PAGE we left to develop overnight, we will incubate the now cleaned biofilm plates with the same supernatants used to PAGE yesterday.

Day 39

Run SDS-PAGE in the morning for the supernatants

(L, E, J and P)# prepare supernatants and run PAGE. Potentially do more if growing in falcon tubes

Day 40

(L, E, J and P)# left secreting overnight - isolate supernatants for SDS-PAGE

Week 9

Day 42

Set Up for TCA

Dilute the 4 Art-175 overnights - 16 conical flasks

Secrete at 18C overnight and TCA ppt tomorrow

Secretion

Prepare 50/100 ml volumes of Ara + cell cultures for purification from supernatant

Determine buffers for purification.

Day 43

Expression

Lysis and His tag purification of P# (DNase)

SDS-PAGE for Nickel column fractions collected Day 42

TCA ppt for overnight secretions of Art-175 constructs - store ppts

TCA ppt for the overnights (secretion for four hours in the afternoon) → Dilute 1/20 in LB + appropriate antibiotic first thing in the morning so that we can start secretion asap

Purification

So far - DNase DsbA and DspB DsbA expressed 4hrs, DNase expressed overnight, Art-175 group expressed 4 hrs and overnight

Setting up secretion for DsbA tagged and DspB that I can run on a column tomorrow - 4 hrs secretion

Western Blot

Art-175 group expressed 4hrs and overnight - ready to PAGE O,K,N,L

Set up overnights for DsbA group - prepare TCA ppts from overnights tomorrow - also need to set up secretion overnight for DsbA group.

Day 44

Expression

Art-175 buffers made up

Run columns for Art-175 group (50ml + 20ml 4hrs secretion with no resuspension buffer) + DNase (resuspension buffer)

Day 45

Expression

MG constructs 1/40 dilution and purify supernatants to run on column

Lysis of cell pellets to run on column

SDS-PAGE for Art-175 group

Week 10

Day 46

Grew up 3x J# DF, and H# MG overnights to 0.6 OD, and followed protocol past NiNTA purification, ready for SDS-PAGE tomorrow.

No bands.

Day 47

Duke (aka king of banter)

Listened to reggae music (you know, Bob Marley, UB40, the classics) whilst eluting L#, M# and O# (all in DeltaFli) through nickel column, ready for SDS-PAGE tomorrow.

Day 49

Western Blot

NB: Duke/Silas when analysing the results, refer to the gels in the Coomassie blue stain - there is some inconsistency as to how lanes were loaded i.e. if a lane was left free between the ladder and the samples

Refer to the Western blotting protocol

Primary Ab stored as 10 uL aliquots in the -20CTo immunostain, dilute by 1/1000 in 10 mL 1% milk powder and PBS (the full 10 uL aliquot in 10ml milk solution). NB: Andrea said that this will probably be too bright - let’s see what she thinks when we come to analyse the data
The PBS and PBS + Tween 20 (Tween 20 is the same as Tween - it’s a typo on the protocol) solutions are on our shelf
The secondary Ab is in the back fridge, top shelf, has a mouse drawn on it. This also requires 1/1000 dilution into 10 mL 1% milk solution (remember to dilute the 5% milk solution with PBS, not water) for the second staining step
Refer to Andrea’s help for the visualisation step

Secrection

Constructs induced to express and pellets frozen - stored in the -80C

Day 50

Western Blotting

Carry on with immunostaining of the blots on the shaker

Carry on with immunostaining of the blots on the shaker
They were washing in PBS overnight - this is not normally required - in the future stick to the timings on the protocol

Immunostain the two blots that have been in milk solution overnight

Biofilms

Week 5

Day 25

Test crystal violet staining of biofilms (using 23/07 tox assay plate)

Protocol.

0.1% w/v crystal violet solution made up by dissolving 0.080g crystal violet in 80mL Milli-Q.

96-well plate was taken out of plate reader at 10am and left on the bench until 4pm.

Contents of plate decanted into biological waste bucket and each well of plate washed with tap water for 4 rinses to remove planktonic cells.

125µL of 0.1% crystal violet solution added to each well, and left to stain for 20 minutes.

Contents of plate decanted and each well rinsed thoroughly again to remove unstained crystal violet.

Plate left to dry overnight.

Comments: Growth curve assay plates are non-ideal for biofilm screening as the shaking incubation of the plates in the plate reader means that biofilms are formed more on the walls of the wells than the bottom of the wells.

Week 6

Day 27

Biofilm Cultivation Using Existing Transformed Strains

Biofilms grown in triplicate wells according to tox assay layout, except the wells with 0.002% final arabinose conc is replaced with 0% arabinose conc (i.e. arabinose replaced with Milli-Q).

Each well has 100uL total volume of 1:100 diluted overnight culture.

Round bottomed plate was used.

Left for incubation on bench for 48 hours.

Day 30

Biofilm Staining

First, measure the OD600nm of the biofilm culture.

Decant and wash the round-bottom well plate that has been incubating at room temperature for 2.5 days (see protocol)

Do CV staining and monitor 500-600nm using whichever machine is available

If biofilms are shown to grow, we will proceed with monitoring biofilm kinetics

Week 7

Day 31

Assays

Various biofilm assays

3 modified E. coli plates and co-culture with wild-type (for testing of biofilm inhibition); 3 wild-type plates for eventual testing of biofilm degradation

N.B.30degC plate (modified E. coli plate) has B6 and B7 swapped around

Day 34

Set Up

Set up conical flask cultures for E# and J# (both strains) to test with wt-E. coli biofilm plates

1:10 dilution with LB amp

1.5 hours initial incubation, 4 hours incubation with 0.2% final conc of arabinose (190µl of 20% arabinose in 19ml of solution), incubation from 12:30 to 4:30pm

OD values after initial incubation:

hisB MG : 1.12, hisB deltaFliC : 0.73, E# MG : 1.1, E# deltaFliC : 0.72, J# MG : 0.74, J# deltaFliC : 1.2

4mL of supernatant isolated from each culture, kept in cold room for addition to E. coli biofilms set up on 03/08

Day 35

Biofilm Assaying

Set up P. putida 96-well biofilm plates and incubate for 48 hours at 30 degC without shaking: P. putida overnight culture diluted 1:100 in a square petri dish in LB Broth (no antibiotics), and 100uL of the diluted culture filled into each well of one tissue culture-treated round bottom-well 96-well plate.

Set up E. coli MG1655 96-well biofilm plates and incubate for 48 hours at room temperature: MG1655 (with pBAD/HisB) diluted 1:100 in a square petri dish in LB Broth supplemented with ampicillin, and 100uL of the diluted culture filled into each well of one tissue culture-treated round bottom-well 96-well plate.

Incubate E. coli biofilm plates that were cleaned yesterday with supernatants from E# and J# that were isolated yesterday.

Wash some overnight cultures of E#, J#, K#, N#, and control deltaFliC with non-antibiotic tryptone broth (same way as Interlab step ii) twice, then grow them in non-antibiotic tryptone broth (1:50 dilution, make 20mL cultures) with a final arabinose concentration of 0.2% for 4 hours. The supernatant here will eventually be used for both secretion assay and biofilm assay/cell-killing assay with P. putida. 4x 1.4mL aliquots of supernatants were obtained from each culture.

Week 8

Day 36

Biofilm Assaying

NB: All we’ve been doing thus far is getting constructed E. coli to grow biofilms (do our constructed cells with ara make biofilms? do cells not secreting grow biofilms? Always do an OD during biofilm prep to make sure you have cells!)

Currently, we also have wild type E. coli growing biofilms to which we add constructed E. coli supernatant

Retrieve plates that have E. coli biofilms being incubated with supernatants from 37 degC incubator, wash and do CV staining to test for biofilm degradation (Leon remember to fill in the layout file for this (dated 07/08). (Raffy, the OD of these plates wasn’t taken thus ii) being done)

Retrieve MG1655-biofilm growth plate (for eventual degradation) from benchtop, take OD600 reading to make sure every well has had cells in them, then decant and wash planktonic cells away. The plate is then ready to be incubated with supernatants.

Retrieve P. putida-biofilm growth plate (for eventual degradation) from 30 degC, take OD600 reading to make sure every well has had cells in them, then decant and wash planktonic cells away. The plate is then ready to be incubated with supernatants (make sure antibiotic free supernatants - the ones in cold room made up on Friday are antibiotic free).

Set up again biofilm viability plates using cultures of our constructed E. coli. Can potentially set up two plates, both of which left on bench (~25degC incubation). (Raffy, is biofilm growth viable?)

Set up biofilm inhibitory plates by culturing P. putida in supernatants of our constructed E. coli, as well as non-constructed E. coli as controls.

Day 37

If visual inspection tells you that P. putida biofilms indeed have formed in the previous 96-well plate, set up two more of them using the test tube of P. putida in the 30 degC incubator (1:100 dilution per well, 100µL of diluted culture per well).

Day 39

We have set up viability (on the bench) and is according to Raffy’s toxicity map 12/08, leave another day to grow.

Set up another viability assay same as yesterday

Also have set up degradation for putida and E. coli - write the map down; stain tomorrow afternoon

Grow some more biofilms to degrade

Biofilm inhibition assays - grow E. coli biofilms in DspB and DNase supernatant

Day 40

E. coli biofilms growing on the bench (set up at 5 on 14/08) - leave until Saturday

Read biofilm degradation - retrieve 96 well plates from incubator. Read OD600, then wash and read OD spectra between 500-600

Use the overnight supernatants to degrade the biofilms growing from 12/08

Set up another inhibition plate

Week 9

Day 42

Set Up

O# and L# O% arabinose growth curve and biofilm viability
Test 1 day old putida biofilm with supernatants
Biofilm viability for E# and J#

NB: *** Take OD reading before you stain the biofilm (just because they don’t form biofilms when you induce expression)

Didn’t overnight putida - overnight tomorrow to test to cell killing assay on - also grow another putida biofilm

Week 11

Day 54

P. putida testing

1 plate of biofilm grown for 1 day

K, L, N, O, HisB, grown at 30°C for 2 hours, then induced with 0.4% ara at 30°C for 4 hours, then left to sit at room temperature for 2 hours

100µL of supernatant from above added to 100µL of stationary culture of P. putida, and 100µL of 1:20 diluted culture which have been left sitting for 4 hours.

Run in clariostar together with toxicity assay

Preliminary observations: Toxicity in H and L as expected

N supernatant displays some extent of P. putida-killing capacity - secretion conditions to be further investigated.

Biofilm Viability Assay

Stationary cultures diluted 1:100 into 200uL Amp-supplemented LB per well.

Incubation at room temperature.

K MG 0.2% ara (1-3)	L MG 0.2% ara (4-6)	N MG 0.2% ara (7-9)	O MG 0.2% ara (10-12)
K MG 0.02% ara (1-3)	L MG 0.02% ara (4-6)	N MG 0.02% ara (7-9)	O MG 0.02% ara (10-12)
K MG 0% ara (1-3)	L MG 0% ara (4-6)	N MG 0% ara (7-9)	O MG 0% ara (10-12)
ctrl MG 0.2% ara (1-3)
ctrl MG 0.02% ara (1-3)
ctrl MG 0% ara (1-3)

Observation: L MG 0.2% and 0.02% shows significantly impeded cell growth (low cell density) - perhaps it would be better to test for biofilm growth by placing them in wells for induction using a mid-log culture (grown without arabinose beforehand).

Biofilm viability assay

1:100 dilution of stationary cultures into 100µL of antibiotic-supplemented LB in each well of round-bottom 96-well plate.

1 plate for MG, 1 plate for RP. Same layout.

Incubation at room temperature for 2 day.

	0.2% ara (1-3)	0.02% ara (4-6)	0% ara (7-9)	10-12
E				HisB 0.2% ara
H				HisB 0.02% ara
J				HisB 0% ara
K
L
N
O
P

Washed and stained biofilm viability assay, but not yet analyzed

Preliminary observation seems to suggest that incubating for one day is not enough to form detectable biofilms.

Over the Weekend

Biofilm degradation assay

Retrieve 48-hour putida biofilm plate from Day 54 - attempt degradation using artilysin supernatants
(on Monday, consult Chris on how to tune Ca2+ concentration and pH of supernatant)

Analyze MG Day 54 biofilms

Cell Killing

Week 8

Day 39

Lyse P, O and H to show that it is being expressed just not secreted - ask Chris or George about sonication.

Fluorescence

Week 6

Day 29

Growth Curve

For the protocol see here.

Plate reader hanged on first run; forgot to turn the incubator back on after resetting the program, so it’s a room temperature growth curve.

Day 30

Assay

Fill black well plate with 100uL of stat phase overnights, same layout as usual, take instantaneous read (fluor and OD) using FluorStar.

Week 8

Day 36

Quorum-Sensing Fluorescence Assay

Wash C* MG1655 and deltaFliC in modified M9 media (+ thiamine supplement) once. Dilute 1:100 and monitor growth in microplate wells.

Week 10

Day 50

Quorum-Sensing Fluorescence

1/20 dilution of C* and pBAD33 in LB + Chl

Incubate until OD ~ 0.6

Notes:

C* is pLsr - GFP
“We are using this part to qualitatively test that pLsr functions as expected in response to AI-2, as well as quantitatively measure the kinetics of gene expression when pLsr is induced by a given amount of AI-2.”
We want to show that GFP expression can be induced by AI-2. To do this, it is necessary to use the supernatant of C* in exponential phase and culture stationary phase bacteria (overnight culture) in the supernatant

Producedure:

Incubate cultures to correct OD
Transfer to a falcon tube and spin down at full spin for 15 mins
Transfer the supernatant to separate falcon tubes

Set up 70-80 cycles (~25-30 hours) OD + GFP

Flow cytometry

Interlab

Week 2

Day 8

Preparation of Interlab Study BioBricks

10µL Milli-Q added to wells 20K, 22A, and 22K in Plate 1, and well 21J in Plate 4 in the iGEM Distribution Kit to resuspend the necessary BioBricks [pSB1C3] to prepare for the Interlab study.

The plates were kept on mild shaking until the afternoon.

Transformation of Interlab Study BioBricks into DH5alpha 1.5

Conduct plating under the filter hood. Add antibiotic to molten agar whilst in bottle, such that the antibiotic (Chl or Amp) is diluted by 1000 times. Then gently mix. Pour just enough agar to cover the surface of the petri dishes. Plates should take ~30mins to set.

Then add volume of E. coli according to protocol, using beads to spread across petri dish.

Day 9

Growth and Culture of Bacteria

Refer to section 1.6 of the protocol guide.

Only one overnight culture each set up for iGEM distros (20K, 22A, 22K, 21J) as it can be rightly assumed that plasmids supplied by iGEM HQ are high purity.

Day 10

Plasmid for PCR Products

Digest aliquots of each of the samples, and run on gel. (Refer to protocol in section 1.2)

From gel, determine the degree of success of these samples.

Week 3

Day 11

Restriction Digest of InterLab Study Plasmids

20K, 22A, 22K:

Component	Volume / µL
Plasmids	10
SpeI	1
PstI-HF	1
Milli-Q Water	6
CutSmart	2

21J: Same as above, but replace SpeI for XbaI.

Incubated for 2hrs at 37℃, then enzymes heat inactivated at 95℃.

Day 12

Restriction Digest of InterLab BioBricks

Gel Photo

Pulse spin
Dephosphorylate 20K, 22A, 22K, BUT NOT 21J
Load dye and run gel

Gel image (transilluminator lens was quite grainy today)

Appropriate bands excised for extraction (I13504 for last lane)

Gel extraction of InterLab BioBricks

340uL of Binding Buffer;

one elution with 50uL for 20K, 22A, 22K; 30uL for 21J

Ligation of InterLab sequences

Insert: digested 21J

Plasmids: 20K, 22A, 22K

Component	Volume/µL
T4 Ligase	1
T4 Buffer	4
Insert	10
Plasmid	10
Milli-Q	26

NB: total reaction volume of 51µL is 1µL more than ideal

NB: total reaction volume of 51uL is 1uL more than ideal

Day 15

Group D

Miniprep plasmid extraction for 20K and 22A tubes.

Nanodrop concentration measurement. Tom noted that most of our concentrations are too low for sequencing (≤100ng/μl) and suggested that we elute using 35μl elution buffer that had been prewarmed in 55℃ water bath and then pass the filtrate through column and spin down again for better yields.

In the afternoon we performed the restriction digests of the plasmid and prepared the gel to be run on Monday for the digest products.

The gel has been left in a tank with buffer and a lid.

Week 4

Day 16

Lychee

Run gel for digested plasmids from 10/7. Check if bands are of correct size, if so, send sample for sequencing

Gel run no.1: Bands overrun into the bottom half of the gel - redo. But 21K[22A] and 21J[22K] seemed to show correct-sized bands → send for sequencing

Did gel extraction for 21J bands from gel but did not obtain a satisfactory concentration of DNA -> restriction digest to obtain 21J tomorrow

21J[22A] and 21J[22K(2)] sent for sequencing

Day 17

Group D

Restriction digest for 20A to get 21J. Religate 21J into plasmid.

Digest the remainder of 10/07 miniprep products and run on gel

Rest of bands are still not giving the right inserts, but 20K seems to give the right size → sent for sequencing on 15/07, and stored in freezer

Day 20

Ria and Lychee

Interlab plasmids 20K, 22A, 22K are ready for transformation.

All 20K, 22A, 22K plasmids used up in transformation into DH5alpha because there didn’t seem to be enough plasmids - need to miniprep a portion of the overnight cultures of these transformed cells to replenish plasmid stock.

Week 5

Day 21

Miniprepping of 19/07 overnight cultures

For interlabs - make frozen stock with 700µL of each culture (add 300µL glycerol to it, vortex, and put in -80degC); miniprep the rest of the cultures to replenish plasmid stocks.

Transformations

Interlab (22A, 22K, 20K) (after miniprep is done)→ MG1655, deltaFliC

Interlab positive and negative controls:

(+) - BBa_I20270 (Kit Plate 3, Well 8P)
(-) - BBa_R0040 (Kit Plate 2, Well 6F)

Resuspend in 10uL Milli-Q, then transform into DH5alpha

Plate Streaking

Frozen stocks of DH5alpha 20K, 22K, 22A also streaked

Day 23

Miniprep of DH5α (+) and (-)

Stocks made - 700µl LB + 300µl 60% glycerol

Nanodop:

	[DNA] (ng/µl)
DH5α (+) 1	39.6
DH5α (+) 2	27.1
DH5α (-) 1	28.5
DH5α (-) 2	33.7

Transformation into FliC and MG1655 - left in incubator overnight

Frozen Stock Preparation

Interlab 20K, 22K, 22A in MG1655 and deltaFliC made into frozen stocks

Day 24

Overnights

Pick colonies and overnights for DH5α (+) and (-) in FliC and MG1655.

Day 25

Frozen Stocks

For (+), (-) in MG1655, deltaFliC

Overnight cultures in M9 Modified Media

(+), (-) DH5alpha, MG1655, deltaFliC from frozen stocks.

20K, 22K, 22A DH5alpha from plate

20K, 22K, 22A MG, FliC from frozen stock

All Chl.

Over the Weekend

Overnight cultures taken out onto the bench after being incubated at 37degC, 225 rpm orbital shaking for 20 hours.

100µL culture loaded into each well for GFP fluorescence and OD600 measurement.

Sodium fluorescein used as standard (absolute units) for fluorescence data to be compared against. 1.66g fluorescein (free acid) dissolved in 5mL pH8.0 Tris HCl to obtain 1M fluorescein solution, neutralized with 5mL 2M NaOH to obtain 10mL of 0.5M sodium fluorescein (NaFluo).

Serial dilution of NaFluo performed for construction of calibration curve.

Week 6

Day 27

Growth curves for InterLab

*note: 20K DH5alpha cultures failed to grow (overnight test tubes very clear)

*see protocol information in the relevant Excel files

Fluorescence Microscopy Trial Run

Single 50mL flask filled with 10mL M9 modified media, inoculated with 1mL overnight culture of 20K MG1655; grown for 2 hours up to OD600 = 0.2.

(note: growth in M9 modified media is very slow compared to that in LB broth, use higher concentrations in the future)

Day 28

Instantaneous Read

GFP fluorescence and OD600

Culturing Strains to Mid-Log

2mL of each overnight culture added to 10mL modified M9 media in 50mL conical flasks, incubated at 37degC, 225 rpm for ~3 hours.

Day 29

Cultivate cells for microscopy

Microscopy done for MG1655 22A, 20K, (22K was already in stat phase despite being OD600 = 0.2 -- cells were short, non-fluorescent); DH5alpha 22A, 20K, 22K

Week 7

Day 31

Microscopy

Dilutions of overnight cultures in 1:5 and preparing slides for microscopy.

Overnights for Interlab cultures were done in LB Chl, and upon 1:5 dilution in M9 stationary phase was achieved within 1.5 hours (validation via OD 0.7-0.9 and microscopy).

Day 32

June

1:5 dilution of interlabs in M9 media, and 45 min of incubation at 37C gives OD value of 0.4 to 0.5. DH5alpha strains showed reasonable fluorescence.

Day 33

June

Set up interlab for microscopy

Day 34

Set Up

Set up conical flask cultures of interlabs for microscopy and flow cytometry.

1:10 dilution in M9 media for 45 minutes gave OD values ranging from 0.27 to 0.35. Only MG1655 cultures were grown and analyzed due to limited number of flasks.

Day 35

Tasks

Tasks (no specific order; grab Leon for 4. and 5. at an appropriate time):

Make new bottle of M9 Modified Media without thiamine inside.
Wash (i.e. spin down 1mL of culture for 3 minutes at full speed, then decant, and resuspend in unmodified M9 salts solution) overnight interlab cultures that were grown in LB, and take instantaneous fluorescence reads and OD600. Repeat as many reads as you like (can reuse the same plate because the wells will be filled up with the same type of cells anyway, just make sure to fully empty them each time) until the 1mL of culture you washed is depleted (just so that we don’t waste cells and the availability of the machine).
Set up growth assay - aliquot M9 Modified Media into smaller bottle and add thiamine and antibiotics appropriately. Wash LB interlab cultures using this supplemented M9 media, and grow in plate reader for appropriate duration. We only have access to the FluorStar overnight today.
Grow up cells of your choice in conical flasks in LB (under 1 hour if 1:5 dilution; if longer growth time desired dilute in 1:20, 1:50, or even 1:100 as necessary). Stop incubation of these cells at OD600 ~ 0.4 - 0.7, then wash with unsupplemented M9 salts to halt growth. These cells can then be used for microscopy and flow cytometry.
Microscopy: remember to book the microscope under the “confocal” sheet outside the door. Also, after completing session, consult Chris for demonstration on how to clean up microscope objective lens.
Flow cytometry: Consult Chris to arrange a time when George is also available, and work out how to use the flow cytometer properly.
Microscopy image analysis: If no one else has a laptop with MATLAB installed, grab Leon to learn microscopy image processing from Chris. Before that, get Leon to copy all the image files we’ve had thus far out of the microscopy PC.

Week 5

Day 36

Tasks

Basically repeat as much of the Day 35 workflow as possible, briefly:

Wash overnight cultures in M9 media (unsupplemented) for single-read fluorescence and OD600
Wash overnight cultures in modified M9 media (+ thiamine supplement) for growth curve
Set up flasks of washed culture (appropriate dilution) in modified M9 media (+ thiamine supplement) for microscopy and flow cytometry.

Day 37

M9 salts haven't arrived, should start interlab from making M9 media. Same as usual, except please do catch GDubz for cytometry.

Wash overnight cultures in M9 media (unsupplemented) for single-read fluorescence and OD600
Wash overnight cultures in modified M9 media (+ thiamine supplement) for growth curve
Set up flasks of washed culture (appropriate dilution) in modified M9 media (+ thiamine supplement) for microscopy and flow cytometry.

Day 38

Focus on MGs

Flow cytometry - 1/25 cultures in at 9.20

Set up for growth curves (only have 2 sets)

Trouble shooting for microscopy

Ask Chris to go through using the microscope

Try a 1/10 dilution

Day 39

Grow falcon tubes - DH5alpha this time → for cytometry and microscopy (1:25 dilution for flask shown to be good for MG)
All cultures overnighted - can do full growth curve

Day 40

Grow falcon tubes - 15x interlab overnights 1:20 dilution into Chl LB, for cytometry and microscopy.

All cultures overnighted - can do full growth curve

Day 41

22 overnights to dilute 1/20 in LB + Chl and culture until mid-log OD ~0.4-0.7. Follow flow cyt protocol
Microscopy with mid-log phase cultures

Look at the InterLab page - Imperial 2014. Analyse the microscopy - Chris to show June or Lychee. Not bothering with the growth curves. Compile the flow cytometry data. Microplates - Leon

Day 42

Finish microscopy

Set up calibration curve with following concentrations of sodium fluorescein:

0.01µM	0.1µM	0.2µM	1µM	2µM	5µM	10µM	20µM

This should give a calibration curve that tallies perfectly with the range of interest for spectrophotometer settings.

Day 43

Analysis of microscopy images for 20K, 22K and 22A in different bacterial strains (DH5ɑ, MG and ΔfliC) using MicrobeTracker.

Prepared overnights for (+)MG, (+)DH5ɑ, (-)DH5ɑ, (+)ΔfliC, (-)ΔfliC to visualise under microscope tomorrow (20/8)

Day 44

(+)MG, (+)DH5ɑ, (-)DH5ɑ, (+)ΔfliC, (-)ΔfliC were visualised on the microscope.

Overnights set up for (+)MG, (+)DH5ɑ, (-)DH5ɑ, (+)ΔfliC, (-)ΔfliC for observing tomorrow

Day 45

Visualised (+)MG, (+)DH5ɑ, (-)DH5ɑ, (+)ΔfliC, (-)ΔfliC

Used MicrobeTracker to identify cells in images and ran GFP mesh analysis on MicrobeTracker

Week 5

Day 47

Microscopy

Visualised 22AΔfliC on the microscope.

Histograms plotted for mean pixel intensity of cells across different strains and the different genes using MicrobeTracker.

Day 48

Mean pixel intensity per cell for each biological replicate calculated using MicrobeTracker. Mean and standard deviations calculated for the different biological replicates.

Flow cytometry data recorded its mean and standard deviation calculated.

Synbiota

Week 7

Day 33

PCRs

pLas RDP F and R - use B gBlocks (157bp) - 72*C
T4 Holin F and R - use D gBlock (672bp) - 72*C
ArtE F and R - use M gBlock (594bp) - 72*C
pLsr F and R - use D gBlock (90bp) - 72*C
LasR F and R - use B gBlock (717bp) - 72*C

PCR conditions: 10-40s per kb recommended. None of the sections are 1kb, and for ease of PCR, I’ve used a 20s extension time for all. If this doesn’t work, we can always try again, but I think shorter than 20s might be too quick… I’ve also used 30 cycles.

PCR products in freezer, ready for gel tomorrow morning. Gel has been poured and will be left in buffer overnight.

Day 34

Gel Photo

Run gel for K# → K* and Synbiota RDP PCR done yesterday

For LasR and pLas, the RDP parts can be PCR-ed from parts already in the iGEM distribution kit instead.

LasR - BBa_C0179: 2015 distro Kit Plate 3, Well 6M (pSB1C3)
pLas - BBa_R0079: 2015 distro Kit Plate 3, Well 5C (pSB1C3)

Transform these tomorrow.

Week 8

Day 37

Synbiota PCR - Art-E from M or T4 holin from D; synbiota pack is in the cold room - they supply their own ladder as well as buffer to dilute primers in. Phusion can be obtained from Chris, and we have our own Phusion HF buffer in the -20deg

Miniprep the overnight cultures for T4Endo, LasR, pLas

Day 38

Continue with Synbiota clean-up protocol (Art-E PCRed yesterday according to protocol and is in the freezer) (Mabel - now given to Leon to run gel)

Day 39

Gel Photo

Carry on with the Art-E synbiota gel in the cold room

No bands - George to repeat PCR

Clean up synbiota digests in freezer (these are plasmids that have been linearised for PCR). PCR according to synbiota protocol with other Synbiota PCRs - in synbiota box in freezer.

Day 40

Carry from on Synbiota PCR - PCR tubes in working shelf of the freezer

Week 9

Day 41

Email Synbiota with gel picture

Re-PCR pLas and pLsr

PCR Art-E from M* if sequence is good

Gel from Friday:

Lane 1 - not sure
Lane 2 - is pLsr - run again to make sure
Lane 3 - LasR (ignore the larger band)
Lane 4 - T4 holin (primers designed to only amplify holin)

Day 43

PCR all synbiota parts again. plsr and t4 holin worked, other two failed. Will keep trying PCR with plas and LasR.

Day 44

PCR plas and LasR again. gel -> still nothing. Ran another PCR using a different protocol, but still got nothing.

Week 10

Day 47

Gel order: Ladder, T4 Holin, pLsr, M*- #, M*- #

Deformed gel - agarose may have been mixed with water instead of TBE

Repeat PCR from very beginning… :( :( :(

Linearise template in 10ul digests

Heat inactivate for 30mins at 95degC before setting up PCR according to RDP protocol

Run on 2% agarose gel - 1g in 50ml, 120V for 30 mins (this is too short - can afford to run longer for better separation)

Gel order: Ladder, pLsr, T4 Holin, Art-E, LasR, pLas, M* - #, M* - #

Excised all bright bands except for Art-E and pLas - repeat PCR tomorrow

Day 48

Gel extraction/cleanup of synbiota and M* - # bands

Synbiota: eluted with 55degC 2 x 50ul TE buffer

M#: eluted with 55 degC 50ul EB buffer

Emailed Synbiota with gel picture and nanodrop values

Repeat PCR of Art-E and pLas, but digest template with 1ul of template regardless of [DNA] in 10ul digest

Template: T4 Endo* and pLas pSB (i.e. the other eppendorf of template to the ones used on 25/08 PCRs)

Also add DMSO to PCR set up, at 5% and 10%.

Day 49

Run gel of Art-E and pLas PCR from Day 48

N.B. 5% or 10% = % of DMSO in PCR set up

D#- *	D#- *	Art-E 5%	Art-E 5%	Art-E 10%	Art-E 10%	pLas 5%	pLas 5%	pLas 10%	pLas 10%

Left ladder: 50bp (from Chris, in synbiota box)

Right ladder: 1kb+ ladder

Farmost right well has loading dye (trying to identify an unnamed eppendorf on bench)

Art-E’s brightest bands are too small - it is ~220bp long when it should be 629bp long. Potentially due to primers being ordered wrong and getting promoter?

Excised both D* bands (880bp, now in freezer next to M#)

Awaiting reply from Patrick for Synbiota

Week 11

Day 52

Patrick replied - reattempt with:

Double the reaction volume for those that worked (200ul rxn)
GC buffer for those that didn’t work

Art-E and pLas: use template from digests done on 26/08

pLsr, T4 Holin and LasR: linearise template in 10ul reactions as on 25/08, with the exception of LasR which needs [template] correction.

Art-E and pLas: GC buffer

pLsr, T4 Holin, LasR: 200ul reaction (will need to divide into 4 wells)

Gel: 2% agarose in TBE, 120V

LasR	LasR	LasR	LasR	T4 Holin	T4 Holin	T4 Holin	T4 Holin
pLsr	pLsr	pLsr	pLsr	pLas (GC)	pLas (GC)	Art-E (GC)	Art-E (GC)

Excised all T4 Holin and pLsr bands - in freezer

Day 53

Cleanup of Synbiota pLsr and T4Holin

Had to re-PCR(used x4 elution buffer and had nanodrop results of pLsr: 16 and T4Holin: 9)…… Sorry Mabel!!!

Waiting for the protocol of next step from Synbiota

Day 54

Proceed with Synbiota RDP Parts Creation Protocol - BsaI digestion Protocol.

BsaI Digestion (2.5hrs)

DNA cleanup

Measuring and adjusting the part concentration

Day 55

Repeat pLas, LasR and Art-E at 55 and 59 degC annealing temperatures

Digested templates from 25/08 (LasR) or 26/08 (Art-E and pLas)

Only do 50ul reaction - if it works then scale up

2% TBE agarose, 120V

LasR 55	pLas 55	ArtE 55	LasR 59	pLas 59	Art-E

LasR works! YAAAAAAAAAAASSSSSSSSSSSSSSSSSSSSS

Scale up after the weekend at 59 degC

Made with love by Oxford iGEM

Plate	Colony	Plate	Colony
C#	1	C#e	1
D#	0	D#e	0
E#	2	E#e	2
H#	0	H#e	3
K#	0	K#e	0
L#	0	L#e	0
M#	0	M#e	0
N#	0	N#e	0
P#	1	P#e	0

Plate	Colony	Plate	Colony
C#	1	C#e	1
D#	0	D#e	0
E#	2	E#e	2
H#	0	H#e	3
K#	0	K#e	0
L#	0	L#e	0
M#	0	M#e	0
N#	0	N#e	0
P#	1	P#e	0

Team:Oxford/Test/Notebook

Notebook

Parts Guide

Parts Guide

Cloning

Week 1

Day 1

Preparation of Stock Solutions

Sequences

Preparation of Reaction Solutions

Polymerase Chain Reaction

Gel Electrophoresis of PCR-Amplified gBlocks

Day 2

Gel Electrophoresis of PCR-Amplified gBlocks (continued from 22/06)

Results

Extraction of DNA (PCR product) from Agarose Gel

Restriction Digest of Extracted PCR product

Restriction Enzyme Digest of the iGEM HQ linearised pSB-1c3

DNA ‘Cleanup’ using EZNA enzymatic reaction kit

DNA Quantification using NanoDrop

Ligation of gBlock Sequences with pSB-1C3 Backbone

Preparation of Competent E. coli Cells

Re-PCR of DNA Sequences

Day 3

Re-PCR of DNA Sequences

Plasmid recovery

Extraction of “sticky” pSB1C3 (recovered from 2014 pSB1C3+abijk) from Gel

Gel weight:

Repeat PCR with higher DNA concentration (for the sequences that previously did not yield clear bands)

Gel extraction of G and J

Gel weights:

Restriction Digest of G and J

NanoDrop Quantification

Ligation

Transforming E. coli cells with Plasmid DNA

Day 4

Growth and Culture of Bacteria

Transformation of J and G (24/06 PCR Product) into competent E. coli cells

Primer Design

Day 5

Plasmid Extraction

Plasmid Quantification

Plasmid Digest

Analysis of Results

Week 2

Day 6

PCR of samples C,D,E,H,J,L and N

Gel electrophoresis of C,D,E,H,J,L and N

EZNA gel extraction of C,D,E,H,J,L and N

Restriction Digest of Gel Extracted C,D,E,H,J,L and N

Recovery of pSB-1C3 vector from 2014 pSB-1C3 + insert

EZNA gel extraction protocol on recovered pSB1c3 vector

Growth and culture of E. coli transformed with 24/06 PCR Product (J and G)

Day 7

From yesterday

Ligation of 29/06 PCR products to pSB-1C3 (1.3)

Plasmid Extraction using miniprep kit (1.7)

Plasmid Quantification: Nanodrop (1.22 Nanodrops are usually done following restriction digest and transformation)

Plasmid Restriction Digest for 24/06 PCR Product 1.2

Gel Electrophoresis of Digested Plasmids from 24/06 PCR Product 1.1

Preparing new primer stock solutions

PCR Amplification of gBlocks Using New Primers

Day 8

Gel Electrophoresis of 30/06 PCR Products

Restriction Digest of 30/06 PCR Products

EcoRI-HF + PstI-HF: C*, D*, H*, L*, M*, N*

BspHI + PstI-HF: E#, K#, N#, L#, H#

BamHI: C#, D#

NcoI, PstI: M#, P#

Plasmid Design

Transformation of 29/06 ligation products (containing C, D, E, H, L, N gBlocks)

Day 9

Plasmid restriction digest - pSB1C3, pBAD33, and pBAD/HisB

Enzymatic Reaction Clean-up - 30/06 PCR Products

Growth and Culture of Bacteria - 29/06 PCR Products

Ligation of 30/06 PCR Products(

Preparation of Competent E. coli DH5alpha

Day 10

Plasmid for 29/06 PCR Products

Preparation of Competent E. coli DH5alpha

EcoRI-HF + PstI-HF: C, D, H, L, M, N

13/07 PCR Batch (E, E DMSO)

PCR of A, B, I, F, K*, A#, B#, F#, I#

Plate	Colony	Plate	Colony
C#	1	C#e	1
D#	0	D#e	0
E#	2	E#e	2
H#	0	H#e	3
K#	0	K#e	0
L#	0	L#e	0
M#	0	M#e	0
N#	0	N#e	0
P#	1	P#e	0