Re-PCR of DNA Sequences

Refer to protocol from 1.0

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

1. Incubate the mix above for 2hrs at 37℃
2. Incubate for 30mins at 95℃ to inactivate restriction enzymes (Inactivation temperature of SpeI is 80℃, and EcoR1-HF is 65℃)
3. Cool and spin (there will be condensation at the top of the eppendorf)
4. Add 1µl CIP (phosphatase) and incubate for 30mins at 37℃
5. Add loading dye
6. Load each DNA on the gel
7. 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

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.

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.

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.

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

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.

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

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:

1. Pulse spin primers
2. Add fresh Milli-Q as above table
3. Leave for 30 mins at 37C in the shaking block (allowing DNA to resuspend)
4. 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

Gel Electrophoresis of 30/06 PCR Products

Protocol 1.2

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.

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.

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.

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.

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

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:

1. Spin down solid
2. The amount of primer is given in y nmol for each tube. Add 10y µL of water to each tube to make 100µM.
3. 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.

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

1. 37℃, 2 hours (heat up another block to 95℃)
2. Heat inactivation, 95℃, 20 minutes
3. Melt, cool, and pour 1% agarose
4. Pulse spin
5. CIP dephosphorylation (1 µL CIP), 37℃, 30 minutes
6. 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)

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 (E*1 or E*2) 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 (J*1 or J*3) 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.

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

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

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#

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

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

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

A# used up the last of the 2014 Q5 MasterMix

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

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

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.

Mini-prep overnighted J*

6 colonies grown overnight

Alterations to protocol:

1. Spin down entire volume of LB broth to ensure all E. coli are used in the extraction.
2. Pulse after spinning down all the E. coli to remove excess LB
3. 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

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#.

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.

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:

1. 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.
2. 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.

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 J*QC, F*, I*, R#, L#

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

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

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 D*IIe, E*1 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.

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.

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

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

Transform

J#QC → DH5alpha - Amp

DsbADNase (from iGEMHQ) → DH5alpha - Chl

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

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

Raffy

PCR C,D,L,M,N#

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

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)

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

Miniprep

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

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

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

George

Miniprep G#

Helen

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

PCR synbiota stuff

• 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.

Cleanup C#, M# digested → ligate

Mabel and James

Set up overnights - add J# to them

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

Kyle

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

Transform C* into MG, FliC

Helen

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.

Transform ligated C#, M# into DH5alpha.

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

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

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*

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

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.

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

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

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

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

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.

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.

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.

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

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.

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

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

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.

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

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

Overnights

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

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.

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)

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.

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

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).

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.

June

Set up interlab for microscopy

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.

Tasks

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

1. Make new bottle of M9 Modified Media without thiamine inside.
2. 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).
3. 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.
4. 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.
5. 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.
6. Flow cytometry: Consult Chris to arrange a time when George is also available, and work out how to use the flow cytometer properly.
7. 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.

Tasks

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

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

Trouble shooting for microscopy

Ask Chris to go through using the microscope

Try a 1/10 dilution

Microscopy

Visualised 22AΔfliC on the microscope.

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