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 » et « 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
1. E/S Digestion of the Biobrick from the original plasmid and reinsertion in the E/S pre-restricted wanted plasmid.
2. Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI, BL21).
3. Add an LB-agar in a petri dish and cultivate the bacteria in rich medium added with antibiotics of interest.
Test the Transformation
1. Run the obtained bacteria culture DNA in a PCR and an electrophoresis gel (qualitative assessment)
2. Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
3. 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”
1. Do an E/S digestion on the first biobrick and an X/P on the second
2. Ligate the two biobricks then insert them into the plasmid
Transforming Bacteria with a new BioBrick
1. E/P Restriction of the biobrick and ligation into a E/P restricted plasmid
2. Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI ou BL21).
3. Pour an LB gel in a petri dish and cultivate the bacteria in an antibiotic rich medium
Test the Transformation
1. Run the obtained bacteria culture DNA in a PCR and pour an electrophoresis gel (qualitative assessment)
2. Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
3. Do an E/P digestion verification to see if the inserted biobricks have the correct size (On only 100ng of plasmids)
4. Send to sequencing
Repeat steps 4, 5, 6 until obtaining the wanted sequence

Protocol #1 : Preparation of competent bacteria cells

  Everything should be done in sterile conditions and on ice (4°C).

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 80mL 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 200 mL of culture:

Preparation of 80 mL of Tbf1 Buffer

KAc 1M	2.4 mL
MnCl2 0.5M	8 mL
KCl 1 M	8 mL
CaCl2 0.1M	8 mL
Gly 80%	15 mL
H2O	38.6 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

Team:Aix-Marseille/Experiments/Protocols

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