2 red colonies (which is weird)
Liquid culture for amplification

We get expected bands but, as for second PCRs of VVD and YN155 fragments, bands 2 time heavier than expected bands appeared

We get an amplification of pDawn independent of the temperature

After the last Gibson reaction we have get red colonies that should correspond to bacteria transformed with pSB1C3-RFP (which should be impossible after the gel migration and purification). So we analyzed the content of the gel extraction and we saw that the tube only had linearized backbone. About pDawn III PCR 2, the migration of 5 µl from the gel extraction of PCR products have shown that there is not a high quantity of DNA material, not enough for a Gibson assembly…

28 July 15

No amplification

DNA mix:
YN155 1: 1,2µl + 1,3µl of MQ water
YN155 2: 1,2µl + 3,2µl of MQ water
VVD1 : 1µl + 2,1µl of MQ water
VVD2 : 1µl + 0,5 µl of MQ water
pSB1C3 : no dilution
mix= 2µl of each

5µl used for the Gibson assembly added to 15µl of master mix
Transformation of 100 µl of competent cells using 5 µl of Gibson product

A total of 12 colonies in both plates containing transformed bacteria
No colony into the negative control
“Biofilm” into the positive control, bacteria are alive and competent

We used the kit from QIAgen to extract the DNA from the gel
Final elution into 50 µL

37°C, 60 min

We get expected bands for our plasmid
No amplification of pDawn

A total of 12 colonies in both plates containing transformed bacteria
No colony into the negative control
“Biofilm” into the positive control, bacteria are alive and competent

We used the kit from QIAgen to extract the DNA from the gel
Final elution into 50 µL

37°C, 60 min

No colonies
We should try again with a plasmid we already transformed
Bacterial survival has to be tested

NEB double digestion: Buffer 2.1
EcoRI:100%, PstI:75%

37°C, 100 min

We get confirmation of the presence of pSB1C3 and our part, but there is still the unknown band. We have cut the heaviest band of the fourth wells to isolate our plasmid from the other one.

Transformation of 100µl of homemade competent cells with 1 µl of pSB1C3 containing a RFP following our protocol and plated onto two agar plates containing chloramphenicol
100 µl of homemade competent cells have been split and plated onto two agar plates, one with chloramphenicol and the other one without antibiotic

Transformation of 50µl of competent cells with 1 µl of pUC19 following our protocol
50µL of competent cells have been used as negative control
Both of them have been plated onto agar plates containing chloramphenicol

NEB double digestion: Buffer 2.1
EcoRI:100%, PstI:75%

37°C, 60 min

We get expected bands at approximatively 2000 bp and 750 bp, but it still an extra band at 1500 bp (already present during the previous electrophoresis). That could correspond to another plasmid.

Aim: second step for pDawn amplification and mutagenesis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn I, II and III

Good amplification of pDawnI and II
The amplification of pDawn 3 is unexpected and cannot be used for the following experiment
About the miniprep of bacteria transformed with the Gibson assembly product: two plasmids have been purified (only one expected); additional checking steps will be lead to characterize these plasmids

QIAquick gel extraction
2 bands of each fragment (pDawnI and II) into 1 column
Concentrate into 30 µl of Elution Buffer

Aim: step for pDawn amplification and mutagenesis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Small amplification of pDawnIII

QIAquick gel extraction
4 bands of pDawnIII into 4 columns (due to high volume of buffer)
Concentrate into 25 µl x4 of Elution Buffer

Massive liquid culture into an Erlenmeyer of 500 ml (100 ml of LB medium) using few ml of pre-liquid culture
Application of the protocol for the production of competent cells 95 aliquots of 100µL

Good bacterial amplification --> miniprep with a MOBio kit (20 ml into 2 columns, final elution with 50 µl for each)
The first PCR to amplify pDawn III has been made by our advisor Samuel Juillot.

Bacteria have grown on the plate; no bacteria into the control
Pre-liquid culture into an Erlenmeyer without selective condition using an isolated colony from the plate; with a control (Erlenmeyer without bacteria)

Aim: first step for pDawn amplification and mutagenesis

After 48h, 2 white shapeless colonies have grown --> Liquid culture (20mL of LB + 20µl of Cam)

Culture of E.coli DH5 alpha from NEB on a non-selective agar plate with a control (plate without bacteria)

Aim: first step for pDawn amplification and mutagenesis

No colonies have grown

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of the Gibson product

Soft bands: it means that some reactions occured

Aim: first step for pDawn amplification and mutagenesis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

No amplification

Aim: first step of our VVD Biobrick

DNA mix:

• YC155: 1µL + 9µL of MQ water
• VVD1: 1µL + 1µL of MQ water
• VVD2: no dilution
• pSB1C3: 3.5µL + 1.5µL of MQ water

3 tubes: - 20µL of competent cells + 5µL of Gibson product - Idem - 20µL of competent cells as negative control
Same protocol as used before but only 200µl of LB has been added to resuspend bacteria. Then the final volume of bacteria which has been plated was 50 µl. 37°C, O/N

Aim: first step for pDawn amplification and mutagenesis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

No amplification

Aim: first step for pDawn amplification and mutagenesis

Aim: first step of our VVD Biobrick

DNA mix:

• YC155: 1µL + 9µL of MQ water
• VVD1: 1µL + 1µL of MQ water
• VVD2: no dilution
• pSB1C3: 3.5µL + 1.5µL of MQ water

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Soft band at 1500 bp, good but not enough

Aim: first step for pDawn amplification and mutagenesis

iGEMTAQ program

Aim: Migration of PCR product

Gel : 1,2 g agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of PCR products YC155, pDawn I, II and III

We get expected bands for YC155, pDawn I and II but there is no amplification for pDawn III
First explanation: human error We should make the reaction again

Aim: purification of fragment from PCR

QIAquick gel extraction
2 bands of each fragment into 1 column
Concentrate into 50 µl of Elution Buffer

iGEMTAQ program

Aim: Migration of PCR product

Gel : 1,2 g agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of PCR products purified YC155 and pDawn III

No amplification of pDawn III
YC155 quantification = 128 ng.µL^-1

NEB double digestion
Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 65min

Aim: check for digestion

Migration of the three digested pSB1C3 samples

QIAquick gel extraction
4 bands (1,175 g) into 1 column
Concentrate into 30 µL of Elution Buffer

Aim: quantification of DNA

Migration of digested pSB1C3 samples

Quantification by Image J: 91 ng.µl^-1

Protocol from MOBIO Kit

• pSB1C3: 20 ml into 4 columns, eluted with 30 µl (pooled together)

Gel: 1,2 g of agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of purified pSB1C3

Quantification of pSB1C3 by Image J = 35 – 40 ng.µL^-1
4 µg (min) of pSB1C3 are requiring for digestion of the plasmid to make a Gibson reaction

• 35 ng x 115 µl (remaining) = 4,025 µg all the remaining 115 µL of pSB1C3 should be digested

11 June 15

Protocol from QIAgen, UltraClean® Standard Mini plasmid Prep Kit

• BBa_I712074
• BBa_K124003

Stock of BBa_I712074 and BBa_K124003 (cf protocol for glycerol stock), -80°C

Migration of purified BBa_I712074 and BBa_K124003

Bands do not correspond exactly to expected weights (supercoiled)

3 plates: 20 mL of LB agar + 20 µL of Cam

Double click tube: 3 mL of LB broth + 3 µL of antibiotic

Survival checking of bacteria after a night spent into a -20°C freezer
Erlenmeyer: culture of bacteria transformed with pSB1C3 coming from glycerol stock; 20 mL of LB broth + 20 µL of Cam

Amplification of bacteria already transformed with BBa_I712074 (Promoter T7) or BBa_K124003 ( Endolysin/Holin)
On petri dish: 20 mL LB Agar + 20µL Amp & 20µL Kan
LB Broth: 2,5 mL of LB + 2,5µL of Amp & 2,5µL of Kan

Miniprep test using liquid culture of Terminator T7 and RBS to check the efficiency of miniprep kit from QIAgen and MOBIO

Aim: Verification of the purification

Migration of miniprep products

Bands have more migrated than expected but it could be due to the folding of circular plasmid. Both kits are efficient.

NEB double digestion
Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Migration of digested T7 and bJun from 29.05.2015 and digested T7 and RBS from 05.06.2015

After digestion we get expected band for T7 terminator and RBS from minipreps of the day

Aim: first step of our VVD Biobrick

Master Mix from University of Freiburg (15µL)
Mix of DNA fragment:

• 1.5 µL pSB1C3
• 1 µL PCR.2 VVD1 (diluted 1/100)
• 2 µL PCR.2 VVD2 (diluted 1/100)
• 1 µL YC155 (diluted 1/10)

Aim: transformation of E.Coli with Gibson Assembly product

20 µL of competent cells + 5 µL of Gibson product
Protocol for bacteria transformation: using 200 µl of LB broth before incubation for 1 h at 37°C and discarding 150 µl of supernatant before plating

Aim: Extraction of the biobrick’s backbone for Gibson Assembly

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Aim: electrophoresis for gel purification

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

We get expected bands
Extraction of the heaviest band of digested pSB1C3 and all the others expected bands for VVD 1, VVD 2, YC155, YN155 1, YN155 2.

Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer

Aim: quantification of ADN to perform Gibson Assembly

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

There is not enough DNA to detect and quantify pSB1C3 and YC155 Try again

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer

No bacterial growth

Aim: check for digestion product from 29.05.2015

No bands for miniprep samples or for digestion products
The miniprep has failed

Results of the liquid culture from 28/05/2015

After 24h of growth:
Terminator T7 bacteria growth strongly into both tubes
bJun bacteria growth into strongly one tube only, the other one is “cloudy”
bFos both tube are “cloudy”
Control negative clear

Stock of Terminator T7 and bJun (cf protocol for glycerol stock), -80°C

Aim: plasmid purification from transformed bacteria

Protocol from QIAgen, UltraClean® Standard
Mini plasmid Prep Kit
Terminator T7: 2 tubes
bJun: 1 tube

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• SpeI: 100%

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Aim: purification of fragment from 28.05.2015

Use of a QIAquick gel extraction kit with the protocol of QIAgen
Gel weight = 270 mg

Aim: first step of our VVD Biobrick

Master Mix from University of Freiburg (15µL)
Mix of DNA fragment:

• 1 µL pSB1C3
• 2 µL PCR.2 VVD1
• 2 µL PCR.2 VVD2
• 2 µL YC155

Aim: transformation of E.Coli with Gibson Assembly product

Incubate at 25°C during 60 hours

Aim: preparation of selective LB agar plate

Add 40 ml of LB agar into a Falcon tube (50ml) + 40µl antibiotic For 20 ml / plate (2 plates)

Antibiotics concentration:

• Ampicilin: 50 µg.ml^-1 diluted 1/1000

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation
100 µl remaining competent cells have been put at -80°C again (loss of efficiency)

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the control plate
Many colonies into the other plate

Aim: Extraction of the biobrick’s backbone for Gibson Assembly

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Aim: check for digestion

1 gel for purification: 110ml TAE 1X + 1,1g agarose + 140µl BET
Sample (purification): 20µl of digestion product+ 2µl of loading dye (6x)
Sample (quantification): 1µl of DNA + 2 µl of loading dye (6x) + 9µl of water MQ
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl loading dye (6x) + 9 µl water MQ
110 V, 45 min

Antibiotics concentration:
Tube 1 & 2: 3 mL LB + 3 µL Cam; colonies from “1µL” plate of Terminator T7 transformation
Tube 3 & 4: 3 mL LB + 3 µL Amp; colonies from “50µL” plate of bFos
Tube 5 & 6: 3 mL LB + 3 µL Amp; colonies from “50µL” plate of bJun
Tube 7 & 8: 3 mL LB
37°C, 120 rpm, O/N

Results of the transformation from 25/05/2015:

After 24h of growth:
No colonies into negative controls Many colonies appears into plates with bFos and bJun transformed bacteria No colonies into plates with pSB1A2 transformed bacteria:

• It could be due to the presence of 2 antibiotics (high environmental pressure)

Plasmid characteristic:

• Part name: BBa_J61100
• Plasmid: pSB1A2
• Part name: BBa_J61100
• Resistance: AK (Amplicilin/Kanamicin)

Punch a hole without removing the foil
Pipette 10µl of water MQ (up and down)
Let sit for 5 minutes to make sure the dried DNA is fully resuspended

Aim: preparation of selective LB agar plate

Add 40 ml of LB agar into a Falcon tube (50ml) + 40µl antibiotic For 20 ml / plate (2 plates)

Antibiotics concentration:

• Ampicilin: 50 µg.ml^-1 diluted 1/1000
• Kanamycin: 50 µg.ml^-1 diluted 1/1000

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the 2 control plates
Many colonies into all the other plates
Colonies from pSB1A2 transformation should exhibit red color

Aim: second step of mutagenesis for YN155

Mix PCR (x8) without primers, DNA and Taq pol
PCR cycle IGEM TAQ program, Tm = 61,3°C

Aim: check for digestion

Remaining gel (120ml TAE 1X + 1,2g agarose + 150µl BET) with 8 wells
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 26 min

We get expected bands with a good intensity meaning DNA have been highly amplified.

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Use of 3 colonies (3 tubes) from pSB1C3 (BBA_B0015) 1µl and pSB1C3 (BBA_B0015) 4µl
3ml LB Broth + 3µl Cam in double click tubes (12ml) + 1 control tube (3ml LB Broth)
37°C, 120rpm, O/N

Aim: amplification of YC155 and first step of mutagenesis for YN155

Mix dNTPs: 2 µl of each dNTP
Mix PCR without primers, plasmids and Taq Pol
PCR cycle IGEM TAQ program

Aim: check for digestion

1 gel: 120ml TAE 1X + 1,2g agarose + 150µl BET
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 25 min

We get expected bands with a good intensity meaning DNA have been highly amplified

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Aim: plasmid amplification

3 plates with 25 ml LB medium + 25 µl of Cam antibiotic / plate
Recover dried DNA sent by iGEM

• Punch a hole without removing the foil (Plate 3, well F3)
• Pipette 10 µl of water MQ (up and down)
• Pipette 10 µl of water MQ (up and down)
• Protocol for bacteria transformation

• E.coli DH5 alpha (NEB5 alpha competent coli)
• Aliquot of 50 µl of competent E.coli / transformation

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min. Do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 100 µl
Plate bacteria and incubate at 37°C, O/N

Results
Control was cloudy as cultures (lack of antibiotics) should be done again
21 May 15

Aim: second step for VVD amplification and mutagenesis

Run of the IGEM TAQ program

Aim: check for digestion

1 gel: 120ml TAE 1X + 1,2g agarose + 160µl BET
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 30 min

We get expected bands about 250 bp meaning DNA have been highly amplified

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Aim: first step for VVD amplification and mutagenesis

Run of the IGEM TAQ program

Aim: check for DNA amplification

Low melting point gel
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x)
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ
110 V, 26 min

We get expected bands about 200 bp with a good intensity meaning DNA have been highly amplified without contamination.

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Aim: second step for VVD amplification and mutagenesis

dNTP preparation (4µL):
1 µl dATP 100 mM
1 µl dCTP 100 mM
1 µl dTTP 100 mM
1 µl dGTP 100 mM
Store at -20°C

Aim: first step for VVD amplification and mutagenesis

dNTP preparation (4µL):
1 µl dATP 100 mM
1 µl dCTP 100 mM
1 µl dTTP 100 mM
1 µl dGTP 100 mM
Store at -20°C

Mix preparation for 5 tubes: 43,5 µl / tube

Results from the liquid cultures: all the cultures have grown

Aim: plasmid purification from transformed bacteria

Protocol from MO BIO, UltraClean® Standard
Mini plasmid Prep Kit l
4 plates with Ampicillin

Dilution of the glycerol in water
Percentage in mass not in volume
Ex: 50% glycerol solution 50 g Glycerol + 50 g water (mQ)
Then filtration with vacuum pump

Aim: store the transformed bacteria for later use

Weight 50 g of pure Glycerol
Weight 50 g of distilled water
Mix and homogenize the solution
Filtration with vacuum pump, then close the bottle under PSM
Under PSM class II: put 1ml of liquid culture in a 1,5 ml sterile tube (pDusk, pDawn, psB1C3, VVD)
Centrifugate 1mL of liquid culture 4000 rpm, 5 min
Remove and discard the supernatant
Under PSM: resuspend with 0,5 ml of LB without antibiotics
Add 0,5 ml of glycerol 50% (25% final)
Homogenize
Transfer in an identified cryotube and store at -80°C

Results observation for the transformed cells culture (in Petri dishes): no colonies for the positive control pUC19, other plates and negative controls are as expected.

Antibiotics concentration:
Use of 3 colonies (3 tubes) from VVD, pDawn, pDusk, pSB1C3 3ml LB Broth + 3µl
Antibiotic in double click tubes (12ml) 37°C, 120rpm, O/N

Aim: preparation of selective LB agar plate

Add 50 ml of LB agar into a Falcon tube (50ml) + 50 µl antibiotic
For 25 ml / plate (2 plates)

Summary:
6 plates with Kanamycin
3 plates with Chloramphenicol
4 plates with Ampicillin

Antibiotics concentration:
Ampicillin: 50 µg.ml^-1 diluted 1/1000
Chloramphenicol: 25 µg.ml^-1 diluted 1/1000
Kanamycin: 50 µg.ml^-1 diluted 1/1000

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the 3 control plates
Many colonies into all the other plates;
Colonies from pSB1C3 transformation should exhibit red color

Aim: prepare media for bacteria culture

Qsp 250 ml distilled water
2 bottles of LB broth and 2 bottles of LB agar (+magnetic stirrer)
Auctoclave 121°C