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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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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.
