Team:KCL UK/Experiments
Experiments
Overview
Step I. Preparation of plasmid pSB1C3 containing BBa_J23100 promoter
We transformed XL1 Blue cells with pSB1C3 containing BBa_J23100 2018 iGEM distribution Kit plate 1 Well 20I and plated on chloramphenicol containing LB agar plates. We inoculated single colony into 10ml LB media containing chloramphenicol antibiotics and grew over night. Plasmids were then isolated and stored at -20℃. We digested some of the plasmid DNA with SpeI and PstI restriction enzymes. The digested plasmid DNA were run on 1% agarose gel and purified. The purified plasmid DNA was stored at -20℃.
Step II. PCR amplification of sRNA without Target Binding Region and restriction enzyme digestion.
We synthesised sRNA constructs without target binding region by PCR amplification by the IDR with KCL_iGEM_6F and KCL_iGEM_6R primers. We ran the constructs on 2% agarose gel and purified them. We digested PCR amplified sRNA constructs with XbalI and PstI restriction enzymes. The sRNA constructs with XbalI and PstI restriction enzymes were purified using electrophoresis on 1% agarose gel and stored at -20℃.
Step III. Constructing pSB1C3 plasmid composed of BBa_J23100 promoter and sRNA with no Target Binding Region
We ligated pSB1C3 plasmid containing BBa_J23100 and digested with SpeI and PstI restriction enzymes, which were prepared in part I, with PCR amplified sRNA constructs, which were digested with XbaI and PstI restriction enzymes, which were prepared in part II. We introduced this ligated plasmid into the XL1Blue cells by doing transformation and plating on chloramphenicol containing LB agar plates. After 16 hours of incubation, we inoculated a singe colony over night in 10ml of LB media. We isolated the plasmids to obtain the DNA. The obtained plasmids were sequenced and the DNA was stored at -20℃.
Step IV. Site directed mutagenesis to create an expression pSB1C3 plasmid composed of BBa_J23100 promoter and sRNA with B0032 RBS Target Binding Region.
In order to achieve site-directed mutagenesis, we used the plasmids created in Part III and forward primers with sRNAs (1_GcvBFor, 2_MicAFor, 3_Spot42For, 4_RybBFor, 5_OmrBFor, 6_RprAFor, 7_RyhBFor, 8_DsrAFor, 9_SqrSFor, 10_MicCFor), reverse primer SDsRNARev and Quick Change II XL Site-directed mutagenesis kit. We did transformation on XL1 Blue cells with chemically competent E.coli and plated on chloramphenicol containing agar plates. We obtained a single colony from the plates and inoculated over night in 10ml of LB media containing chloramphenicol. The obtained plasmids were isolated and sequenced using BBa_G00100 VF2 sequencing primer. The DNA was stored at -20℃.
Step V. Plasmid pSB1C3 containing BBa_J23100 promoter digestion with EcoRI-PstI restriction enzyme
We digested pSB1C3 plasmid containing BBa_J23100 promoter isolated in part I with EcoRI-PstI restriction enzymes and ran gel electrophoresis on 1% agarose gel. We purified the plasmids and stored them at -20℃.
Step VI. sRNA with BBa_J23100 promoter and B0034 RBS Target Binding Region PCR amplification and restriction enzyme digestion.
We PCRed amplified sRNA constructs containing BBa_J23100 promoter B0034 Ribosomal Binding Site Target Binding Region and sRNA scaffolds synthesised by the IDT with KCL_iGEM_6F and KCL_iGEM_6R primers. We did gel electrophoresis with the amplified PCR constructs on 2% agarose gel and purified them. We digested the PCR amplified sRNA constructs with EcoRI-PstI restriction enzymes. We ran gel electrophoresis of digested sRNA constructs on 2% agarose gel and purified them for ligation.
Step VII. Constructing pSB1C3 plasmid composed of BBa_J23100 promoter and sRNA with BB034 Ribosomal Binding Site Target Binding Region.
We ligated pSB1C3 plasmid containing BBa_J23100 and digested with EcoRI-PstI restriction enzymes, which were prepared in part V, with PCR amplified sRNA constructs digested with EcoRI-PstI restriction enzymes, which were prepared in part VI. We transformed XL1Blue and plated the cells on chloramphenicol containing LB agar plates. The transformed XL1 Blue cells were incubated at 37℃ over night. We inoculated a single colony in 10ml LB media over night. We then isolated the plasmids, sequenced the plasmids and stored DNA at -20℃.
Step VIII. Prepare psB1C3 plasmid containing promoter, RBS and GFP constructs for subcloning.
We transformed XL1 Blue E.coli with pSB1C3 plasmid containing BBa_K608011 Biobrick (J23100 promoter, B0032 RBS and GFP) (iGEM 2019 distribution Kit plate 1 Well 5M) and pSB1C3 plasmid containing BBa_K608010 Biobrick (J23100 promoter, B0034 RBS and GFP) (iGEM 2019 distribution Kit plate 1 well 5K). We plated the cells on chloramphenicol containing LB agar plates. We grew them over night at 37℃. We inoculated single colony into 10ml LB media containing chloramphenicol and grew overnight. The plasmids from the colonies were isolated and stored at -20℃. We digested some of the plasmids DNA with EcoR-PstI restriction enzymes and did gel electrophoresis on 2% agarose gel. We cut the smaller bands and purified to use them for ligation. We stored the plasmids DNA at -20℃.
Step IX. Prepare pSB4K5 plasmid.
We transformed XL1 Blue E.coli with pSB4K5 plasmid containing BBa_J04450 Biobrick (iGEM 2019 distribution Kit Plate 6 Well 6G) and plated on Kanamycin containing LB agar plates and grew overnight at 37℃. On the following day, we inoculated single colony into 10ml LB media containing kanamycin and grew overnight. We isolated plasmid and stored at -20℃. We digested some of the plasmid DNA with EcoRI-PstI restriction enzymes and carried out gel electrophoresis on 1% agarose gel. We cut the larger bands and purified to use them for ligation. We stored the plasmids DNA at -20℃.
Step X. Subclone promoter, RBS and GFP constructs from pSB1C3 plasmid backbone into pSB4K5 plasmid backbone.
We ligated pSB4K5 plasmid backbone, which is digested with EcoRI-PstI restriction enzymes that were prepared in step XI with EcoRI-PstI restriction enzymes digest BioBrick BBa_K608011 and BBa_K608010 that were prepared in step VIII. We transformed XL1 Blue and plated on kanamycin containing LB agar plates. Then we inoculated single colony overnight in 10ml LB media and isolated plasmids. We sequenced plasmids using BBa_G0010 VF2 sequencing primer and stored DNA at -20℃.
Step XI. E.coli cell analysis.
We co-trasnfected XL1Blue E.coli with pSB1Cs plasmid containing appropriate sRNA and pSB4K5 plasmid containing GFP and plated on LB agar plates containing both chloramphenicol and kanamycin antibiotics. The plates were left to grow overnight. We performed 1/10 dilution on overnight cultures into fresh LB media with both chloramphenicol and kanamycin antibiotics and grew at 37℃ taking 1ml samples at 0, 1, 2, 3, 4, and 5 hour intervals. The samples were stored on ice. We aliquoted 200μl of each sample into 96 well black plate with clear bottom and took measurements of OD 600 and fluorescence (GFP filters 485nm (ex) and 540 nm (em)) using 96 well plate reader.
