Team:ULB-Brussels/Projet/Material

==Material and Methods==

==Material and Methods==

Here is the plasmid we built in order to insert the hfsG and hfsH genes :

Here is the plasmid we built in order to insert the hfsG and hfsH genes :

To increase the amount of plasmidic material, we performed transformations with DG1 E. coli competent cells from Delphi Genetics[3]. Its genotype is the following one: mcrA (mrr-hsdRMS-mcrBC, modification-, restriction-) 80lacZM15 lacX74 recA1 araD139 (ara-leu)7697 galU galK rpsL endA1 nupG . For the full protocol see the StabyExpress TM T7 Kit Manual, “transformation using chemically competent cells”[4] .

To increase the amount of plasmidic material, we performed transformations with DG1 E. coli competent cells from Delphi Genetics[3]. Its genotype is the following one: mcrA (mrr-hsdRMS-mcrBC, modification-, restriction-) 80lacZM15 lacX74 recA1 araD139 (ara-leu)7697 galU galK rpsL endA1 nupG . For the full protocol see the StabyExpress TM T7 Kit Manual, “transformation using chemically competent cells”[4] .

All the ligations were achieved in the Assembly standard 10[5].

All the ligations were achieved in the Assembly standard 10[5].

We followed the assembly protocol recommended by New England BioLabs®[6]. The first ligation concerns promoter, RBS and RFP. We cut the promoter plasmid with the restriction enzymes EcoR1 and Spe1. We cut the RBS+RFP plasmid with EcoR1 and Xba1. This one was dephosphorylated to prevent it from self-ligating without any inserts. Seeing that both plasmids are ampicillin resistant, we cut the promoter plasmid with AflIII and ScaI, This will prevent any promoter plasmid of being able to be transformed. At this stage we have promoter, RBS and RFP in the RBS-RFP ampicillin resistant plasmid. (FIGURE2)  

We followed the assembly protocol recommended by New England BioLabs®[6]. The first ligation concerns promoter, RBS and RFP. We cut the promoter plasmid with the restriction enzymes EcoR1 and Spe1. We cut the RBS+RFP plasmid with EcoR1 and Xba1. This one was dephosphorylated to prevent it from self-ligating without any inserts. Seeing that both plasmids are ampicillin resistant, we cut the promoter plasmid with AflIII and ScaI, This will prevent any promoter plasmid of being able to be transformed. At this stage we have promoter, RBS and RFP in the RBS-RFP ampicillin resistant plasmid. (FIGURE2)  

Finally we assembled the first and the second construction to obtain the final plasmid.(FIGURE 1)

Finally we assembled the first and the second construction to obtain the final plasmid.(FIGURE 1)