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Team:Aix-Marseille/Notebook ChassisConstruct
From 2014.igem.org
Notebook: Chassis Construction
Week 7 : 08/11/2014 ➡ 08/17/2014
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We have control if the insert of FRT-Kan-FRT is in the gene sdaBC and CheA by PCR.
# DNA Template Primers Name Weight of amplified DNA 180 Genomic DNA of W3110 CheA_up
CheA_down1700 181 CheA- CheA_up
CheA_down1500 182 Genomic DNA of W3110 sdaBC_up
sdaBC_down3100 183 sdaBC- KanR sdaBC_up
sdaBC_down1500 184 sdaBC- KanR + pCP20 sdaBC_up
sdaBC_down1500 CheA- control is unsuccessful, we see a weight of amplification for #180 and #181 of 1700bp the gene FRT-Kan-FRT isn't insert in cheA.
>SdaBC- control have a problem we don't have amplification for #182 to #184.
Results not shown.
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We made a PCR with new oligo to control mutants.
# DNA Template Primers Name Weight of amplified DNA 185 Genomic DNA of W3110 CheA_check_up
CheA_check_down1700 186 CheA- CheA_check_up
CheA_check_down1500 187 Genomic DNA of W3110 sdaBC_check_up
sdaBC_check_down3100 188 sdaBC- KanR sdaBC_check_up
sdaBC_check_down1500 189 sdaBC- KanR + pCP20 sdaBC_check_up
sdaBC_check_down1500 190 Genomic DNA of W3110 FRT_up
sdaBC_check_down0 191 sdaBC- KanR FRT_up
sdaBC_check_down1600 192 sdaBC- KanR + pCP20 FRT_up
sdaBC_check_down1600 Second control of CheA- show us the gene FRT-Kan-FRT isn't in the gene cheA
SdaBC control have an other problem with amplification
Results not shown.
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We made a gradient PCR 55° to 65° with oligo sdaBC_checkdo/FRTup and W3110 sdaBC- strain and W3110 Wild type.
No amplification shown by the electrophoresis for all these PCR.
Results not shown
Week 8 : 08/18/2014 ➡ 08/24/2014
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We made Q5 PCR with oligo 1-2 and 3-4 on pKD4 template for prepare new transformation sdaBC and cheA respectively #293 and #294. After we supress the template pKD4 with
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We purified PCR products #293 thanks to Macherey Nagel PCR Clean up Kit.
293 22.65 ng.mL-1 -
We made 2 new PCR on transformed strains. With specific hybridation temperature.
# DNA Template Primers Name Weight of amplified DNA Hybridation Temperature 300A CheA- CheA_check_up
CheA_check_down1500 55°C 301A sdaBC- KanR FRT_up
sdaBC_check_down1600 50°C
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We made a PCR with new oligo to control mutants.
# DNA Template Primers Name Weight of amplified DNA 185 Genomic DNA of W3110 CheA_check_up
CheA_check_down1700 186 CheA- CheA_check_up
CheA_check_down1500 187 Genomic DNA of W3110 sdaBC_check_up
sdaBC_check_down3100 188 sdaBC- KanR sdaBC_check_up
sdaBC_check_down1500 189 sdaBC- KanR + pCP20 sdaBC_check_up
sdaBC_check_down1500 190 Genomic DNA of W3110 FRT_up
sdaBC_check_down0 191 sdaBC- KanR FRT_up
sdaBC_check_down1600 192 sdaBC- KanR + pCP20 FRT_up
sdaBC_check_down1600 
After the electrophoresis, we can confirm The strain sdaBC- KanR is successful. We select 3 strains with electrophoresis control
C.23 and C.33 are contaminated. So we select C.43, C.53 and C.63.
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We prepare seven starter of W3110 sdaBC- KanR strain with Kan50.
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We realize day 7 of Lambda Red protocol on W3110 sdaBC- pCP20 strain.
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We realize day 8 of Lambda Red protocol on W3110 sdaBC- strain.
Week 9 : 08/25/2014 ➡ 08/31/2014
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We realize day 9 of Lambda Red protocol on W3110 sdaBC- strain, and day 2 of Lambda Red protocol on W3110 sdaBC- strain with #294 PCR product
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We realize day 3 of Lambda Red protocol on W3110 sdaBC- strain CheA- KanR.
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Check the construction 200(2) (RBS B0034 + RFP E1010) by digestion.
7µL E1010 (control) (500ng)
36µL H2O
5µ NEB Buffer 2.1
1µL XbaI
1µL EcoRI
2µL 200(2) (500ng)
36µL H2O
5µ NEB Buffer 2.1
1µL XbaI
1µL EcoRIIncubation 53' at 37°C. Then, inactivation of enzymes 25' at 80°C.
It is difficult to see if 200(2) possesses the RBS. The plasmid 200(2) is sent to sequencing and it's OK.
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We realize day 4 of Lambda Red protocol on W3110 sdaBC- strain CheA- KanR. strain.
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We realize day 5 of Lambda Red protocol on W3110 sdaBC- CheA- KanR strain.
