Protocols
Protocol #0 : iGEM General Protocol
- Obtaining Biobricks
- EITHER on IDT →
- Order on IDT when the sequences are not too long (chemical synthesis).
- The received biobricks are lyophilized, they must be resuspended in water (cf protocole).
- OR by PCR →
- When sequences are too long and costly to order on IDT, or when a sequence must be added to an existing biobrick :
- Order primers
- synthesize sequence by PCR (cf protocole Q5)
- add the suffixes and prefixes during the synthesis « E, X » and « S, P » (E=EcoRI, X=XbaI, S=SpeI, P=PstI) and homologue sequences to plasmids
- Do a SLIC (cf protocole) to insert the wanted sequence in the wanted plasmid
- the Biobrick is obtained
- Transform the Bacteria with the Biobrick
- E/S Digestion of the Biobrick from the original plasmid and reinsertion in the E/S pre-restricted wanted plasmid.
- Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI, BL21).
- Add an LB-agar in a petri dish and cultivate the bacteria in rich medium added with antibiotics of interest.
- Test the Transformation
- Run the obtained bacteria culture DNA in a PCR and an electrophoresis gel (qualitative assessment)
- Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
- Do an E/P digestion verification to see if the inserted biobricks have the correct size (On only 100ng of plasmids)
- Assemble two biobricks
- “Digestion protocol BioBrick Assembly Kit” et “Ligation protocol BioBrick Assembly”
- Do an E/S digestion on the first biobrick and an X/P on the second
- Ligate the two biobricks then insert them into the plasmid
- Transforming Bacteria with a new BioBrick
- E/P Restriction of the biobrick and ligation into a E/P restricted plasmid
- Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI ou BL21).
- Pour an LB gel in a petri dish and cultivate the bacteria in an antibiotic rich medium
- Test the Transformation
- Run the obtained bacteria culture DNA in a PCR and pour an electrophoresis gel (qualitative assessment)
- Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
- Do an E/P digestion verification to see if the inserted biobricks have the correct size (On only 100ng of plasmids)
- Send to sequencing
- Repeat steps 4, 5, 6 until obtaining the wanted sequence
Protocol #1 : Preparation of competent bacteria cells
Note : Everything should be done in sterile conditions and on ice (4°C).
For 100ml of competent cells
- Grow a culture of bacteria in LB medium until it reaches OD600 = 0.5.
- While it grows, prepare Tbf1 and Tbf2 buffers.
- Centrifuge cells for 10 minutes at 3500g at 4°C
- Resuspend the pellet slowly in 50mL of Tfb1 buffer.
- Centrifuge 5 min at 3500g at 4°C.
- Resuspend the pellet in 8 mL of Tbf2 buffer.
- Incubate 15 min in ice.
- Aliquot 200µL of cell suspension in sterile eppendorf tubes and conserve it at -80°C.
Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers
For 100 mL of culture:
Preparation of 50 mL of Tbf1 Buffer
| KAc 1M
|
1.5 mL
|
| MnCl2 0.5M
|
5 mL
|
| KCl 1 M
|
5 mL
|
| CaCl2 0.1M
|
5 mL
|
| Gly 80%
|
0.93 mL
|
| H2O
|
32.56 mL
|
Preparation of 8 mL of Tbf2 Buffer
| NaMOPS 0.2M
|
400 µL
|
| CaCl2 0.1M
|
6 mL
|
| KCl 1 M
|
8 mL
|
| Gly 80%
|
1.5 mL
|
| KCl 1M
|
80 µL
|
| H2O
|
500 µL
|
Protocol #2 : Transformations
Plasmids transformation
- Add 20 ng of plasmid to 100 μL of competent cells thawed in ice
- Incubate 30-45 min in ice
- Thermal shock : put tubes in the Thermomixer at 42°C for 2 min
- Incubate 5 min in ice
- Add 900 μL {of LB
- Incubate 1 hour at 37°C with agitation
- Spread 100 μL on LB limp (with antibiotic)
Ligation transformation
- Add 20 ng of ligation product to 100 μL of competent cells thawed in ice
- Incubate 30-45 min in ice
- Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
- Incubate 5 min in ice
- Add 900 μL of LB
- Incubate 1 hour at 37°C with agitation
- Centrifuge 5 min at 5000 rpm
- Eliminate 850 μL of medium
- Spread 150 μL on LB limp (with antibiotic)
To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL
Protocol #3 : Cloning protocol for IDT sequences
Resuspension
Resuspend gBlocks Gene fragment to a final concentration of 10 ng/µL in TE or AE (elution buffer).
The tube contain 1000 ng of gBlocks Gene fragment so you have to add 100µL of AE
Mix well, a vortex shall be used
Digestion E/P
1. Digest 100 ng in 50µL
| component
|
50 µL of reaction
|
| DNA
|
10 µL
|
| Buffer 2 (10X)
|
5 µL
|
| BSA (100X)
|
0.5 µL
|
| H2O
|
33 µL
|
| EcoRI
|
1 µL
|
| PstI
|
1 µL
|
2. Incubate for 45 min at 37°C
3. Incubate 20 min at 80°C (inactivation of restriction enzyme)
Ligation
1. Ligate 50 ng vector with a 3X molar excess of gBlocks Gene fragments in fresh T4 DNA ligase buffer diluted to 1X and 400 U T4 DNA ligase for a total volume of 20 µL
| component
|
20 µL of reaction
|
| PSBIC3 (or other vector) at 12.5 ng/µL
|
2 µL
|
| insert digested (at 37.5 ng/µL)
|
12 µL
|
| T4 Buffer
|
2 µL
|
| T4 ligase 400 U
|
1 µL
|
| H2O
|
3 µL
|
2. Incubate 2h at room temperature
Transformation
- Add 10 µL of ligation’s product to 100 µL of competent cells thawed in ice (TGI cells)
- Incubate 45 min in ice
- Thermal shock: put tubes in the thermomixer at 42°C during 2 min
- Incubate 5 min in ice
- Add 900 µL of LB
- Incubate 1 hour at 37°C with agitation
- Centrifuge 5 min at 5000 rpm
- Eliminate 850 µL of supernatant
- Suspend the pellet in the 150µL of remaining medium
- Spread 150 µL on LB limp (with antibiotic)
To make negative control, follow the same procedure but without add plasmids and spread 300 µL
Protocol #4 : Digestion and ligation for verification and BioBrick Assembly
Digestion (verification) protocol
| DNA
|
Between 50 and 100 ng
|
| EcoRI-HF
|
0.2 µL
|
| PstI
|
0.2 µL
|
| 10X NEBuffer 2
|
2 µL
|
| H2O
|
QS 20 µL
|
Incubate all digest reactions at 37°C for 1 hour and then add 3 µL of SES 4X and migrate 30 min at 150V on a 1% agarose gel.
Digestion protocol BioBrick Assembly Kit
Upstream part :
| Upstream part plasmid
|
500 ng
|
| EcoRI-HF
|
1 µL
|
| SpeI
|
1 µL
|
| 10X NEBuffer 2
|
5 µL
|
| H2O
|
To 50 µL
|
Downstream part, optional, for 3A strategy
| Downstream part plasmid
|
500 ng
|
| XbaI
|
1 µL
|
| PstI
|
1 µL
|
| 10X NEBuffer 2
|
5 µL
|
| H2O
|
To 50 µL
|
Destination plasmid :
| Destination plasmid
|
500 ng
|
| EcoRI-HF
|
1 µL
|
| PstI
|
1 µL
|
| DpnI
|
1 µL
|
| 10X NEBuffer 2
|
5 µL
|
| 100X BSA
|
0.5 µL
|
| H2O
|
To 50 µL
|
Incubate the three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.
Ligation protocol BioBrick Assembly
| Upstream part digestion
|
2 µL
|
| Downstream part digestion
|
2 µL
|
| Destination plasmid digestion
|
2 µL
|
| 10X T4 DNA ligase buffer
|
2 µL
|
| T4 DNA ligase
|
1 µL
|
| H2O
|
11 µL
|
Incubate at RT for 1 hour.
#Protocol 5 : Generalised transduction using phage P1
Step1 : P1 lysat on the given strain
From a overnight starter, (3mL in 2YT medium + appropriated antibiotiques) from the strand holding the transduct mutation, make a 1/100 dilution in 3mL 2YT medium and add 150µL of Cacl2 0.1M ( without antibiotique corresponding of the transduct allele).
Incubate at 37°C (under agitation) until obtening a DO600=0.5-1 (more 0.5)
Add 150µL of a P1 lysat wt
Incubate at 37°C without agitation (water bath) during 20min
Incubate at 37°C under agitation during 3 or 4h (even overnight)
Divide the culture in 2 eppendorfs of 2mL (=2*1.5mL)
Add 150µL chloroform then vortex
Centrifuge 10min at 4°C and 6000rpm
Transfer 1mL supernatant into 2 news eppendorf of 2mL
Add 10µL chloroform (under a fumehood) in each eppendorf, vortex and keep at 4°C
(option: to increase the titration level, you can do a second cycle)
Step2 : Transduction P1
The eve, prepare a culture of the recipient strain in 3mL 2YT medium (+eventual appropriated antibiotique)
The D-day, start a culture from the recipient strain (1/50 dilution) in 3mL 2YT + 150µL of CaCl2 0.1M
( prepare the LB-citrate 5mM requiered and LBagar- appropriated AB + citrate 2mM petri dishes)
Incubate until reaching a DO600 = 1 or + (2.10^6 bact/mL)
Prepare 3 tubes eppendorf of 2mL (1 control with 500µL cells, 1 with 10µL P1 + 500µL cells, 1 with 100µL P1 + 500µL cells)
Infect during 20min at 37°C (heat wuthout agitation)
Add 1mL steril LB-citrate 5mM and heat at 37°C during 50min under agitation (700-750rpm)
Centrifuge 5min at 5000rpm (RT)
Remove supernatant sterilely with a P1000 and resuspend in 1mL steril LB-citrate 5mM
Spread on LBagar- appropried AB + 2mM citrate
Incubate ON at 37°C
Step3 : Clones isolation
Isolate clones on petri dishes (approprieted AB + citrate 2mM)
Incubate at 37°C
Step 4 : PCR screen and transplantation
Test clones by PCR
Transplant the good clones on LB-AB appropriate petri dishes
