Team:IONIS Paris/Notebook
Plasmid digestion (pSB1C3)
Aim: Extraction of the biobrick’s backbone for Gibson Assembly
| Tube | pSBC3 |
|---|---|
| Buffer 2.1 | |
| Plasmid pSB1C3 | |
| Enzyme EcoRI | |
| Enzyme PstI | |
NEB double digestion Buffer 2.1:
- EcoRI: 100%
- PstI: 75%
37°C, 1h10
Storage: -20°C
Electrophoresis
Aim: electrophoresis for gel purification
Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2
Expected results
Results
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.
Gel Purification
| VVD 1 | |
VVD 2 | |
|||
| YN155 1 | |
YN155 2 | |
|||
| YC155 | |
pSB1C3 | |
Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer
Electrophoresis for quantification
Aim: quantification of ADN to perform Gibson Assembly
Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2
Expected results
Results
There is not enough DNA to detect and quantify pSB1C3 and YC155 Try again
Electrophoresis for quantification - 2nd
Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2
Expected results
Results
| Fragments | Concentration (ng.µL-1) | Lenght | Molecular weight (g.mol-1) | Molarity (mol.L-1) | 1/ratio | Dilution factor | Molarity after dilution |
|---|---|---|---|---|---|---|---|
| pSB1C3 | |
0.000000000642 | 0.000000000642 | ||||
| VVD1 | |
0.000000096698 | 0.000000000642 | ||||
| VVD2 | |
0.000000047103 | 0.000000000642 | ||||
| YC155 | |
0.000000009631 | 0.000000000642 | ||||
| YN155.1 | |
0.000000064805 | 0.000000000642 | ||||
| YN155.2 | |
0.000000114117 | 0.000000000642 |
Liquid culture
| Plasmid | |
Plasmid | Antibiotic | |||
|---|---|---|---|---|---|---|
| Terminator T7 | |
bFos | |
|||
| RBS | |
bJun | |
|||
| Control Negative | |
Control Negative | |
Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer
4 June 15
Results from 29.05.2015
Results of transformation with Gibson assembly product
No bacterial growth
Electrophoresis of digestion product
Aim: check for digestion product from 29.05.2015
Expected results
Results
No bands for miniprep samples or for digestion products
The miniprep has failed
2 June 15
Liquid culture results
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
Glycerol stock
Stock of Terminator T7 and bJun (cf protocol for glycerol stock), -80°C
Miniprep
Aim: plasmid purification from transformed bacteria
Protocol from QIAgen, UltraClean® Standard
Mini plasmid Prep Kit
Terminator T7: 2 tubes
bJun: 1 tube
Plasmid digestion (Terminator T7 & bJun)
| Tube | Terminator T7 | Control negative |
|---|---|---|
| Water | |
|
| Buffer 2.1 | |
|
| DNA | |
|
| Enzyme EcoRI | |
|
| Enzyme SpeI | |
|
NEB double digestion Buffer 2.1:
- EcoRI: 100%
- SpeI: 100%
| Tube | bJun | Control negative |
|---|---|---|
| Water | |
|
| Buffer 2.1 | |
|
| DNA | |
|
| Enzyme EcoRI | |
|
| Enzyme PstI | |
|
NEB double digestion Buffer 2.1:
- EcoRI: 100%
- PstI: 75%
37°C, 1h10
Storage: -20°C
Gel Purification
Aim: purification of fragment from 28.05.2015
Use of a QIAquick gel extraction kit with the protocol of QIAgen
Gel weight = 270 mg
Gibson Assembly
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
Put immediately into a hot bath at 50°C for 1 hour
Transformation
Aim: transformation of E.Coli with Gibson Assembly product
| Plasmid | Plate |
|---|---|
| 3 µL of plasmid | 20 µL on plate |
| 50 µL on plate | |
| 6 µL of plasmid | 20 µL on plate |
| 50 µL on plate | |
| Control negative | 100 µL on plate |
Incubate at 25°C during 60 hours
29 May 15
Amplification of pSB1A2 (part BBa_J61100) into E.coli DH5 alpha
Plate preparation
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)
| Plasmid | Antibiotic | Number of plate | Remarks |
|---|---|---|---|
| pSB1A2 | |||
| Ampicillin | 50 µl of transformed cells | ||
| 20 µl of transformed cells | |||
| Control negative | |||
| Amplicilin | 50 µl of transformed cells |
Antibiotics concentration:
- Ampicilin: 50 µg.ml-1 diluted 1/1000
Cell transformation
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)
| Tube n° | Reagent added | Volume (µl) |
|---|---|---|
| pSB1A2 | |
|
| Control negative | |
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
Plasmid digestion (pSB1C3)
Aim: Extraction of the biobrick’s backbone for Gibson Assembly
| Tube | pSBC3 |
|---|---|
| Water | |
| Buffer 2.1 | |
| Plasmid pSB1C3 | |
| Enzyme EcoRI | |
| Enzyme PstI | |
NEB double digestion Buffer 2.1:
- EcoRI: 100%
- PstI: 75%
37°C, 1h10
Storage: -20°C
Electrophoresis
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
Expected results
Results
Liquid culture
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
28 May 15
Results from cell tranformation
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)
26 May 15
RBS BioBricks
Recover dried DNA sent by iGEM (part BBa_J61100)
Plasmid characteristic:
- Part name: BBa_J61100
- Plasmid: pSB1A2
- Part name: BBa_J61100
- Resistance: AK (Amplicilin/Kanamicin)
Punch a hole without removing the foilRecovered plasmid have been put into a 50µl eppendorf tube called “BBa_J61100, RBS”
Pipette 10µl of water MQ (up and down)
Let sit for 5 minutes to make sure the dried DNA is fully resuspended
Amplification of bFos, bJun, pSB1A2 (part BBa_J61100) into E.coli DH5 alpha
Plate preparation
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)
| Plasmid | Antibiotic | Number of plate | Remarks |
|---|---|---|---|
| bFos | |||
| Amplicillin | 50 µl of transformed cells | ||
| 100 µl of transformed cells | |||
| bJun | |||
| Amplicillin | 50 µl of transformed cells | ||
| 100 µl of transformed cells | |||
| pSB1A2 | |||
| Ampicillin + Kanamycin | 50 µl of transformed cells | ||
| 100 µl of transformed cells | |||
| Control negative | |||
| Amplicilin | 50 µl of transformed cells | ||
| Kanamycin | 50 µl of transformed cells |
Antibiotics concentration:
- Ampicilin: 50 µg.ml-1 diluted 1/1000
- Kanamycin: 50 µg.ml-1 diluted 1/1000
Cell transformation
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
| Tube n° | Reagent added | Volume (µl) |
|---|---|---|
| bFos | |
|
| bJun | |
|
| pSB1A2 | |
|
| Control negative | |
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
25 May 15
PCR VVD BioBricks
PCR.2 Nter (YN155)
Aim: second step of mutagenesis for YN155
| Component | PCR.2 YN155 I (x2) | PCR.2 YN155 II (x2) | Control - YN155 I (x2) | Control - YN155 II |
|---|---|---|---|---|
| Primer Fwd YN155 1 | ||||
| Primer Rev YN155 3 | ||||
| Primer Fwd YN155 4 | ||||
| Primer Rev YN155 2 | ||||
| PCR.1, YN155 I | ||||
| PCR.1, YN155 II | ||||
| Taq Pol |
Mix PCR (x8) without primers, DNA and Taq pol
PCR cycle IGEM TAQ program, Tm = 61,3°C
Electrophoresis
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
Expected results
Results
We get expected bands with a good intensity meaning DNA have been highly amplified.
Purification of PRC product
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
At the end of the purification, we get 2 tubes: “PCR2, YN I, 22/05/2015”, “PCR2, YN II, 22/05/2015”
Stored at -20°C.
Liquid culture
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
22 May 15
PCR VVD BioBricks
PCR.1 YFP Cter (YC155) & Nter (YN155)
Aim: amplification of YC155 and first step of mutagenesis for YN155
| Component | PCR YC155 (x2) | PCR.1 YN155 I (x2) | PCR.1 YN155 II (x2) | Control - YC155 | Control - YN155 |
|---|---|---|---|---|---|
| Primer Fwd YC155 | |||||
| Primer Rev YC155 | |||||
| Primer Fwd YN155 I | |||||
| Primer Rev YN155 I | |||||
| Primer Fwd YN155 II | |||||
| Primer Rev YN155 II | |||||
| Plasmid pBiFC-bFos | |||||
| Plasmid pBiFC-bJun | |||||
| Taq Pol |
Mix dNTPs: 2 µl of each dNTP
Mix PCR without primers, plasmids and Taq Pol
PCR cycle IGEM TAQ program
Electrophoresis
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
Expected results
Results
We get expected bands with a good intensity meaning DNA have been highly amplified
Purification of PRC product
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
At the end of the purification, we get 3 tubes: “PCR1, YC155, 21/05/2015”, “PCR1, YN155 I, 21/05/2015”, “PCR1, YN155 II, 21/05/2015”
Stored at -20°C.
Backbone & Terminator
Transformation pSB1C3 – double T7 terminator (BBa_B0015)
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
Cell transformation
- E.coli DH5 alpha (NEB5 alpha competent coli)
- Aliquot of 50 µl of competent E.coli / transformation
| Tube n° | Reagent added | Volume (µl) |
|---|---|---|
| |
||
| |
||
| |
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
PCR VVD BioBricks
PCR.2 VVD - 2nd part
Aim: second step for VVD amplification and mutagenesis
| Component | PCR.2 VVD 1 (x2) | PCR.2 VVD 2 (x2) | Control negative | Control negative |
|---|---|---|---|---|
| DNA Mix "PCR.1 VVD1" | ||||
| DNA Mix "PCR.1 VVD2" | ||||
| Taq Pol |
Electrophoresis
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
Expected results
Results
We get expected bands about 250 bp meaning DNA have been highly amplified
Purification of PRC product
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
At the end of the purification, we get 1 tube “PCR2, VVD1, 13/05/2015” and another one “PCR2, VVD2, 13/05/2015”
Stored at -20°C.
13 May 15
PCR VVD BioBricks
PCR.1 VVD - 2nd part
Aim: first step for VVD amplification and mutagenesis
| Component | PCR.1 VVD 1 (x2) | PCR.1 VVD 2 (x2) |
|---|---|---|
| PCR mix | ||
| Primer VVD Fwd 1 | ||
| Primer VVD Rev 1 | ||
| Primer VVD Fwd 2 | ||
| Primer VVD Rev 2 | ||
| Water | ||
| Plasmid VVD | ||
| Taq Pol |
Electrophoresis
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
Expected results
Results
We get expected bands about 200 bp with a good intensity meaning DNA have been highly amplified without contamination.
Purification of PRC product
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
At the end of the purification, we get 1 tube “PCR1, VVD1, 07/05/2015” and another one “PCR1, VVD2, 07/05/2015”
Stored at -20°C.
PCR.2 VVD
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
| Component | PCR.2 VVD 1 (x2) | PCR.2 VVD 2 (x2) | Control negative (x2) |
|---|---|---|---|
| Primer VVD Fwd 1 | |||
| Primer VVD Rev 3 | |||
| Primer VVD Fwd 4 | |||
| Primer VVD Rev 2 | |||
| Water | |||
| Buffer RB | |||
| dNTPs | |||
| Mg2+ 50mM |
7 May 15
PCR VVD BioBricks
PCR.1 VVD
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
| T°C | Time | Cycle | |
|---|---|---|---|
| Initial denaturation | |||
| Denaturation | |||
| Annealing | |||
| Extension | |||
| Final extension | |||
| Hold |
| MQ water | 182,5 µl |
| Mg2+ 50 mM | 7,5 µl |
| Buffer RB | 25 µl |
| dNTPs | 2,5 µl |
Mix preparation for 5 tubes: 43,5 µl / tube
6 May 15
Results
Results from the liquid cultures: all the cultures have grown
Miniprep
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
Glycerol preparation & Glycerol Stock
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
1 April 15
Results
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.
Liquid culture
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
31 March 15
Amplification of pDusk, pDawn, pSB1C3, VVD, pUC19 into E.coli DH5 alpha
Plate preparation
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)
| Plasmid | Antibiotic | Number of plate | Remarks |
|---|---|---|---|
| pDusk | |||
| Kanamycin | 50 µl of transformed cells | ||
| 100 µl of transformed cells | |||
| 50 µl of competent cells (neg control) | |||
| pDawn | |||
| Kanamycin | 50 µl of transformed cells | ||
| 100 µl of transformed cells | |||
| 50 µl of competent cells (neg control) | |||
| pSB1C3 | |||
| Chloramphenicol | 50 µl of transformed cells | ||
| 100 µl of transformed cells | |||
| 50 µl of competent cells (neg control) | |||
| VVD | |||
| Amplicilin | 50 µl of transformed cells | ||
| 100 µl of transformed cells | |||
| 50 µl of competent cells (neg control) | |||
| pUC19 | Amplicilin | 50 µl of transformed cells |
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
Cell transformation
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
| Tube n° | Reagent added | Volume (µl) | Tube n° | Reagent added | Volume (µl) | ||
|---|---|---|---|---|---|---|---|
| pDusk | |
pSB1C3 | |
||||
| pDawn | |
(Amp control) | |
||||
| VVD | |
(Kan control) | |
||||
| pUC19 | |
(Cam control) | |
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
30 March 15
Preparation of media
Aim: prepare media for bacteria culture
| LB Broth | LB Agar |
|---|---|
| 2.5g tryptone | 2.5g tryptone |
| 2.5g NaCl | 2.5g NaCl |
| 1.25g yeast extract | 1.25g yeast extract |
| 5.0g agar |
Qsp 250 ml distilled water
2 bottles of LB broth and 2 bottles of LB agar (+magnetic stirrer)
Auctoclave 121°C
28 March 15
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