Team:Aix-Marseille/Experiments/Protocols

Protocols

Protocol #0 : iGEM General Protocol

  1. Obtaining Biobricks
    • EITHER on IDT →
    • µOrder 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
  2. 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.
  3. 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)
  4. 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
  5. 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
  6. 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
  7. 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).

  1. Grow a culture of bacteria in LB medium until it reaches OD600 = 0.5.
    • While it grows, prepare Tbf1 and Tbf2 buffers.
  2. Centrifuge cells for 10 minutes at 3500g at 4°C
  3. Resuspend the pellet slowly in 80mL of Tfb1 buffer.
  4. Centrifuge 5 min at 3500g at 4°C.
  5. Resuspend the pellet in 8 mL of Tbf2 buffer.
  6. Incubate 15 min in ice.
  7. 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

  1. Add 20 ng {of plasmid to 100 μL of competent cells thawed in ice
  2. Incubate 30-45 min in ice
  3. Thermal shock : put tubes in the Thermomixer at 42°C for 2 min
  4. Incubate 5 min in ice
  5. Add 900 μL {of LB
  6. Incubate 1 hour at 37°C with agitation
  7. Spread 100 μL on LB limp (with antibiotic)

Ligation transformation

  1. Add 20 ng of ligation product to 100 μL of competent cells thawed in ice
  2. Incubate 30-45 min in ice
  3. Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
  4. Incubate 5 min in ice
  5. Add 900 μL of LB
  6. Incubate 1 hour at 37°C with agitation
  7. Centrifuge 5 min at 5000 rpm
  8. Eliminate 850 μL of medium
  9. Spread 150 μL on LB limp (with antibiotic)

To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL