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Team:ULB-Brussels/Projet/Material
From 2009.igem.org
Material and Methods
Here is the plasmid we built in order to insert the hfsG and hfsH genes :
PHOTO1
The following bricks come from the BioBricks partsregistry[1]
We chose a lactose inductible promoter so that we could easily control the expression of the genes of interest. This promoter is the BBa_R0011 brick (located in the pSB1A2 ampicillin resistant plasmid) The RBS (ribosome binding site) and the RFP bricks are located on the same plasmid (BBa_K093005). RFP is a reporter gene. If the glue is not released (or we cannot detect it), we will know the genes are expressed. We chose a commonly used terminator, namely BBa_B0015 (located on the pSB1AK3 kanamycin resistant plasmid). The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell) The final plasmid pSB1AK3 is ampicillin and kanamycin resistant. Seeing the importance of hfsG ans hfsH in our project, they were optimized and synthesized1. Their sequences have been optimized for two reasons:
- First they are compatible with all assembly standards.
- Secondly the use of codon is not the same in Caulobacter crescentus and in E. coli. This could limit the rate of translation of our genes.
The hfsG and hsfH sequences show the restriction sites and the optimized codons. (ANNEXE OPTIMIZATION)
Transformation: 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-) 80lacZM15 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] .
Ligations: 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)
PHOTO2
We transformed DG1 E.coli competent cells with this construction. The culture was left overnight (37°C) on ampicillin resistant medium. We selected the appropriated colonies and put them back at 37°C in liquid medium. We proceeded with the extraction of the plasmid using the GenElute Plasmid Miniprep Kit[7]. In order to verify our construction, we cut upstream (EcoR1) and downstream (Pst1) of the Promoter+RBS+RFP. Then we proceeded with the electrophoresis of the digested product. We tested the construction by adding IPTG to a sample and this one was analyzed by fluorescent microscopy.
The second ligation concerns hfsG, hfsH and the terminator. Seeing that both hfsG and hfsH plasmids were ampicillin resistant and that we did not have appropriate enzymes to cut one of them, we chose to ligate hfsH and the terminator (which have different antibiotic resistances). The terminator plasmid is the destination plasmid and hfsH is the insert. The selection was made with kanamycine (following the above-cited protocol). After that, hfsG was added to this construction. At this stage, we have hfsG, hfsH and terminator in the terminator kanamycin resistant plasmid which is our final destination plasmid . (FIGURE3)
PHOTO3
Finally we assembled the first and the second construction to obtain the final plasmid.(FIGURE 1)
