Team:Oxford/Test/Notebook

Notebook

Key

*

A - pSB-1C3 LasR Holin

B - pSB-1C3 LasR sfGFP

C - pSB-1C3 Lsr sfGFP

D - pSB-1C3 Lsr Holin

E - pSB-1C3 DNase DsbA

a

F - pSB-1C3 DspB YebF

G - pSB-1C3 DspB

H - pSB-1C3 MccS

I - pSB-1C3 DspB Fla

J - pSB-1C3 DspB DsbA

K - pSB-1C3 Art-175 DsbA

L - pSB-1C3 Art-175 YebF

M - pSB-1C3 Art-E

N - pSB-1C3 Art-175 Fla

#

A - pBAD 33 LasR Holin

B - pBAD 33 LasR sfGFP

C - pBAD 33 Lsr sfGFP

D - pBAD 33 Lsr Holin

E - pBAD HisB DNase DsbA

F - pBAD HisB QC DspB YebF

G - pBAD HisB QC DspB

H - pBAD HisB MccS

I - pBAD HisB QC DspB Fla

J - pBAD HisB QC DspB DsbA

K - pBAD HisB Art-175 DsbA

L - pBAD HisB Art-175 YebF

M - pBAD HisB Art-E

N - pBAD HisB Art-175 Fla

O - pBAD HisB Art-175

P - pBAD HisB DNase

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 Finderwas 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 NEBCloner. 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

  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

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

  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

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:

  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.

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
  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)

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 (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.

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:

  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

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:

  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.

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 & FliC - E#, H#, N#, P#

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:

  1. 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
  2. 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):

  1. 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)
  2. 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

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

  1. Pulse spin
  2. Dephosphorylate 20K, 22A, 22K, BUT NOT 21J
  3. 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

Overview

Week 3

Day 13

Group A

D* ligated 07/07, to transform today.

C*, D*, H*, M* 07/07 miniprep products to be digested and gelled. Likely get to sequence D* and M* to pocket two more BioBricks.

Dig out the 03/07 miniprep products to send E*2 and J* (not 2 or 6) for sequencing.

“stubborn” PCRs - A*, B*, F*, I*, K*

Transformed and plated the following (for colony formation overnight):

Amp selected: G#, H#, K#, L#, 3.1P#, N#, J#, 3.1O#, E#, O#, #P

Chl selected: 3.1D#, D#, C# and the 3 interlab plasmids.

Somehow we failed to plate one of the C# samples (hence there is only one above). In addition, we found that 20K#e and C#e had been swapped during plating (i.e. the plate labelled 20K#e actually contains C#e and vice versa).

Group B

C#, D# (two sets each) ligated 07/07, to transform today.

C# 07/07 miniprep product to be digested and gelled. If gelling successful, ask Chris which are the pBAD forward and reverse primers used for sequencing.

“stubborn” PCRs - A#, B#

Helen: familiarize with entire workflow by reading Raffy’s newly-made protocols.

Group C

(E,G,H,J,K,L,N,O,P)# ligated 07/07, to transform today.

(E,H,P)# 07/07 miniprep product to be digested and gelledIf gelling successful, ask Chris which are the pBAD forward and reverse primers used for sequencing.

“stubborn” PCRs - I#, F#? Doesn’t matter anyway - DspB-containing sequences can’t be transformed into pBAD/HisB directly; need to “import” from Group A once they are ready to QuikChange (site-directed mutagenesis) G* and J*

Group D

21J ligated into 20K, 22A, 22K - transform today

Day 15

Gel Photo

10/07 PCR Batch 1 - Normal PCR of D*, E*, J*, D#, M#, O#

Attempted with both 2014 and 2015 Q5 Mastermix to test whether the 2014 stock is still working

*second elution was done with 20uL elution buffer to maximize concentration

Restriction Digests

Restriction digest for D*(2014) was done using second elution product. For everything else, the first elution product was used 50µL digest reaction mixtures made using corresponding enzymes (Master Table) according to protocol sheets

Digested mix had their enzymes heat inactivated, and bagged in the -20degC freezer

10/07 PCR Batch II

Defrosted Gel

E* PCRed normally

E* PCRed with 1.26uL DMSO (water correspondingly reduced to 7.74µL

J*4 (completed construct, labelled 30/06 on tube) quikchange PCRed

E*, E*DMSO, J*4 QC, and digested G*6 (see below) run on gel (error - after quikchange-PCRing the DNA is supposed to be incubated with DpnI to destroy methylated plasmids)

(error - freezing large block of gel causes it to crystallize, ruining the bands → E*, E*DMSO, J*4 QC unverifiable)

Miscellaneous

10µL of G*6 (completed construct, labelled 30/06 on tube) was also digested using EcoRI, as the sequencing data apparently shows that there is an extra insert flanked by EcoRI sites on both ends. We hope to digest it out and isolate the correct (linearized) plasmid on a gel)

06/07 PCR Batch

Miniprep of overnight cultures

(C,D,J,K,L,N,G,P,O)# miniprepped according to EZNA protocol

Test restriction digest

Extracted plasmids digested with appropriate enzymes according to Master Table for 1 hour. Gel poured and awaiting electrophoresis on 13/07

Restriction digests carried out for C,D,GJ,K,L,N,O,P #,PBad33,20K,22a,22K using the appropriate restriction digests, nanodrop data was collected from each sample and its repeats also. Once digested gel prep was carried out for each of the samples with 2µl of buffer and 0.5µl of restriction enzyme to give a total volume of 20µl. 10µl of this was then transferred to wells in the gel and ran

Gradient PCR of stubborn sequences

Gel Photo

Outlook

Week 3

Day 13

Going beyond trying to clone the unsuccessful sequences.

Group A - If J sequence successful, can probably QuikChange G and J tomorrow. Time to also look at submission to BioBrick Registry for successful sequences, etc.

Group B - If C# sequencing successful, it is essentially ready. Time to consider: transform to MG1655 (is this the LuxS(-) strain?) → how to isolate AI-2 to induce pLsr → design fluorescence experiment (collaborate with Group D in designing, probably).

Group C - E# is DsbA DNase. If sequencing successful, time to look at how UCL 2012 assayed this part. H# is MccS. For starters, maybe we can see if transforming the part into MG1655 kills our cells…P# is plain DNase. Look at how to experiment on protein isolation through the His tag?

Group D - look at the iGEM website for InterLab. What are the experimental requirements (species of E. coli, etc?)

What Next?

For us, the Oxford iGEM team, it has been a long, hard and very rewarding summer. We wish we could continue our project further, but now, time is our greatest enemy. We hope that our research will pave the way for other groups, both within and without iGEM, to take the reins in the battle against catheter-associated UTIs, and the wider assault on antibiotic resistance.

We hope this page will be a useful guide to teams (in particular UK teams) with medically-orientated projects, who intend to progress from laboratory work into clinical trials. We have also written a brief guide to the process of patenting, and how this would apply to our project in the future.

Preclinical stage

Like most iGEM teams with projects in the Health and Medicine track, we have spent the summer developing our project in the preclinical stage. This is the 'laboratory' stage, where the different components of the project are constructed, tested, and optimised, without the use of human volunteers. The first goal is to discover if the team's invention is feasible; past that point, the preclinical goal is to improve the invention to point the point at which its efficacy and safety of use is maximised.

Progression to clinical trials

Extensive preclinical (lab) data is needed to support the use of the treatment on humans in clinical trials. As described above, the researchers must do their best to minimise the level of risk associated with the treatment, whilst maximising its efficacy, through lab models.

Review bodies

Any group wishing to trial their treatment on volunteers in the UK must abide by the Medicines for Human Use (Clinical Trials) Regulations of 2004. To abide by these terms, researchers must be granted a clinical trial authorisation (CTA) by the Medicines and Healthcare Products Regulatory Agency (MHRA). For a CTA application to be granted, the treatment must be reviewed and certified by a number of different review bodies, where each body scrutinises a different aspect of the project. We will use our project as an example here.

Most medically-orientated projects require approval from a Research Ethics Committee (REC). The committee makes sure that the volunteers have been well briefed (by the researchers) with regards to the trial, including; the possible benefits and risks of the treatment, the goals of the treatment, and who they should contact if they have any further questions. There are several RECs, found across the UK, who deal with clinical trial authorisations. There are several borough-based RECs in London - the iGEM team would most likely apply to one of these bodies.

As our treatment involves the use of a novel device (our 'custom catheter'), we need to apply to the MHRA, for a Notice of No Objection, to review the safety of the device. If granted, the notice would allow the device to be used in volunteers.

Potentially, we may attempt to model the effect our bacterial proteins have on human cells. To get hold of human tissue, we would need a license from the Human Tissue Authority (HTA). This includes extraction of tissue from a cadaver and a live participant.

In addition to the above approvals, we would need an organisation, such as a hospital or research institute, to host our trials. There are several funding bodies to which we could apply; however, it would be ideal to have funding from the NHS, as this is where we would most probably intend to host our trials - this is called Management Permission. Ideally, we would like our trials to be hosted by the NHS! At the time of our Management Permission application, we would most likely apply to the NHS for funding as well.

Clinical trials

We would be very pleased if our project managed to get this far! However, there would still be a long way to go before our product could go on the market. The clinical trials are split into 3 phases, with each phase more rigorous than the last.

Phase I

Phase I trials, also known as 'first-in-human' trials, present the first time a treatment is tested on human volunteers; as a result, there is an unavoidable element of risk involved. A typical phase I trial might involve 10 or fewer healthy student volunteers. However, due to the nature of our treatment, the likely candidates for our phase I trials would be people who already require the use of a catheter, but potentially not those who have a urinary tract infection (UTI).

This phase is primarily a risk assessment, so we would be looking for any signs of immune response to the introduction of our bacteria and their secreted proteins; in particular DNase and Dispersin B. In addition, we may be looking to see what range of protein dosages the volunteers might be able to tolerate before they experience significant side effects or discomfort.

Phase II

If the volunteers from phase I show few side effects and the treatment is deemed safe enough, then the trials can proceed to phase II. These involve up to approximately 100 volunteers. This time, all these people selected should be ill, ie have a CAUTI, and the results of their treatment (reduction in the biofilm size) against the best current treatment; in our case this is antibiotic treatment.

Phase III

If the results of the phase II trials suggest that the new treatment could be better than the current treatment, then the trials can proceed to phase III. As this is the last stage, the organisation hosting the trial must be sure, on a statistical level, that the new treatment is significantly better than the currently-administered treatment, for it to pass. Phase III trials therefore involve a very large group of ill volunteers, sometimes greater than 1000 people.

Marketing license

If solUTIon passed phase III, then we could officially license our treatment, and release it onto the market! Wouldn't that be swell?

Phase IV (optional)

This is an additional phase which is sometimes implemented; however it is not always required! Phase IV 'trials' are slightly different to the previous three, as they monitor the treatment success on real patients, ie after a marketing license has been granted. As the treatment has already been licensed by this point, phase IV simply involves analysis of data from patients receiving the treatment to check its safety and efficacy.

Patenting

What is a patent?

A patent is a license, given to an inventor, which prevents any competitor from making, using or selling their invention in a certain territory for a specific time frame. The 'territory' of the patent may be a country, or even an entire continent, such as Europe. The patent 'time frame' is usually 20 years.

What is the point of a patent?

Patenting is essential to the healthy progression of scientific research; why would companies want to invest millions of dollars into an invention, only for it to be stolen by a competitor when completed? Therefore, patenting gives companies a chance to earn back the money they invest in their inventions. However, the patent holder may, during these 20 years, permit certain parties to make use of their product for a license fee.

What can you patent?

In synthetic biology, any organism (aside from a human) which has been genetically modified, and hence gives rise to "a new assembly of chemicals", can be patented. This includes novel bacterial strains, as well as transgenic plants and animals. Evidently, humans cannot be patented for ethical reasons.

However, there are additional factors that must be satisfied; for an invention to be applicable for patenting it must be both of the following:

  1. Novel - the invention you present should not already be part of the 'state of the art' (basically anything published in a paper, presented at a conference, or anything freely accessible to the scientific community).
  2. Inventive - ie the invention should not be 'obvious' to those skilled in the field. It is often difficult to define if an invention is 'obvious'; however, several 'obvious' inventions can arise from, for example, the correlation of two previously-unlinked scientific papers.

Can Oxford iGEM apply for a patent?

There are a number of aspects to our project which are unpatentable. For example, the BioBricks we have constructed cannot be patented; they have been submitted to The Registry of Standard Biological Parts ('The Registry' for short) as part of our open source agreement. The Registry is iGEM's alternative to patenting; it allows for easy, open distribution of the many hundreds of BioBricks that now exist. In addition, EU law states that methods of therapy, treatment and diagnosis cannot be patented, to avoid conflict with doctors.

However, the device used within the method may be patented. Our method of tackling CAUTIs rests heavily upon the design of our custom-catheter, which is both novel and inventive; we therefore intend to apply for a patent on this device.