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Team:Aix-Marseille/Notebook 09
From 2014.igem.org
Notebook: September
Week 10 : 09/01/2014 ➡ 09/07/2014
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Construction of stability epitopes
The main idea is to use the primers 57-58 and 59 as matrices in order to synthesize and amplify the stability epitope. The PCR uses the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 55°C, the elongation time is 5", the number of cycle is 25.
Mix:
- 5µL OiGEM-57 or 58 or 59
- 2,5µL OiGEM-60
- 2,5µL OiGEM-44
- 25µL Q5 2x Mix
- 15µL H2O
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Clean-up of these PCR and elution with 30µL H2O at 70°C.
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PCR cheA-kan-cheA using the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 60°C, the elongation time is 1’30”.
Mix:
- 1µL pKD4 vector
- 2,5µL primer 3
- 2,5µL primer 4
- 2,5µL dNTP
- 10µL Q5 buffer
- qsp 50µL H2O
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Construction of stability epitopes
Digestion ES of PCR products of the stability epitopes and of piGEM-01.13 (pSB1C3-BBa_J04450).
Mix:
- 10µL K10512-06 or 07 or 08 or 5µL piGEM-01.13
- 1µL EcoRI
- 1µL SpeI
- 5µL Buffer 2.1
- qsp 50µL of water
Incubation 60' at 37°C.
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Then, for K10512-06 or 07 or 08, inactivation of enzymes 20' at 80°C.
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Clean-up of piGEM-01.13 and elution with 30µL of water. Add 1µL of SAP (Promega) + 2µL of SAP buffer (dephosphorylation). Incubation 30' at 37°C, then Inactivation of enzyme 20' at 65°C.
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Ligation K10512-06 or 07 or 08 with piGEM-01.13.
Mix:
- 2µL piGEM-01.13
- 4µL K10512-06 or 07 or 08
- 1µL T4 ligase 10U/µL
- 2µL T4 ligase buffer
- 11µL H2O
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Transformation of 90µL of E.coli TG1 competent cells with 10µL of ligation. The selective medium is LB-Cm 30µg/mL.
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Clean-up of the previous PCR. Elution in 45µL of water.
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Electroporation of W3110 ∆sdaBC electrocompetent cells from Aimeric (clone 43)
40µL cells + 10µL PCR
40µL cells + 0,5µL pKT25 (negative control)
==> Spread bacteria on LB-Kan
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Many clones have grown on the negative control but no clone has grown for the cheA mutant. So cells are competent but they don’t express the recombinase.
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Streak out W3110 ∆sdaBC 43 and 63 from Aimeric on LB and LB-Kan
Week 11 : 09/08/2014 ➡ 09/14/2014
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Construction of stability epitopes
Test 5 clones of each transformation (with pSB1C3- K10512-06 or 07 or 08) by PCR. The temperature of annealing is 55°C, the elongation time is 1'
Mix:
- 2µL OiGEM-60
- 2µL OiGEM-44
- 4µL dNTP
- 1µL DMSO
- 2µL buffer
- 0,5µL Taq polymérase
- 8,5µL H2O
All clones are good except the K10512-08-2.
The pSB1C3-K10512-06-1, pSB1C3-K10512-07-1 and pSB1C3-K10512-08-1 are sent to sequencing. They are OK.
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The bacteria are sensitive to kanamicyn.
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Repreparation of W3110 ∆sdaBC 43 electrocompetent cells (see Lambda Red mutant construction protocol) and restart from this step (day 1).
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The clones obtained were KanR and ApS ==> OK
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The clones are pooled 3 by 3 to be tested by PCR with primers 52 and 53.
The pools 1 and 2 seemed to contain mutants (1786bp).
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Restart PCR with primers 52 and 53 on the isolated clones from pools 1 and 2.
The clones 1a, 2a and 2b seemed good, they are kept at -80°C. So the W3110 ∆sdaBC ∆cheA is OK.
Week 13 : 09/22/2014 ➡ 09/28/2014
Objective: Test the functionality of our RelA part.
The pSB1K3-relA plasmid constructed by the iGEM-AMU team is expected to be able to rescue the phenotype of a MG1655∆relA mutant grown on SMG plates, a medium known to induce a strong amino acid depletion in the cells (Rudd et al.,1985) that is toxic to a ∆relA mutant.
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Day 1:
Isolated clones were restreaked on SMG plates supplemented with 50 uM Kanamycin and 0.5mM IPTG. The plates were incubated at 37°C during 48 hrs.
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Day 2:
Electrocompetent MG1655∆relA (EB421 strain, Wahl et al. 2011) cells were electroporated with either plasmid pSB1K3-RFP as a control or pSB1K3-relA.
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Day 4:
As expected, we observed that the relA construct was able to complement the MG1655∆relA strain, as seen on the following figure.
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SMG plate composition:
M9 salts 1X MgSO4 1mM CaCl2 0.1mM VitB1 0.5µg/ml Glucose 0.2% Serine 1mM Methionine 1mM Glycine 1mM Bactoagar 15g/L References:
- Rudd KE, Bochner BR, Cashel M, Roth JR. Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression. J Bacteriol. ;163(2):534-42, 1985.
- Wahl A, My L, Dumoulin R, Sturgis JN, Bouveret E.Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol. 80(5):1260-75, 2011.
