Team:London Biohackspace/lab-book
The BioBrick encoding the (S. cerevisiae codon-optimised) Miraculin protein coding sequence will be synthesized and ligated into a pSB1C3 plasmid prior to submission to the iGEM registry. Once this part has been synthesized, we aim to use the SureVector expression vector assembly kit to create a plasmid capable of expressing the Miraculin protein. In order to achieve this we will need to PCR amplify the coding sequence from the pSB1C3 plasmid to create a sequence containing the required SureVector overlap sequences. This DNA fragment can then be used as our gene of interest during the SureVector plasmid assembly process. The assembled expression vector will also contain a yeast autonomous replication sequence (yARS) as well as a LEU2 auxotrophic marker to allow for expression in Leucine deficient strains of S. cerevisiae. Expression of the gene will be regulated through the use of the S. cerevisiae X-Gal Galactose inducible promoter provided with the SureVector kit. The SureVector expression plasmid also contains a His-tagging sequence that we can use to characterise Miraclin expression once a yeast strain has be transformed with the plasmid. The DNA sequence encoding the protein coding sequences for the genes CrtE, CrtB and CrtI that are required for lycopene biosynthesis will be synthesized as a single multi cis-tronic sequence with a SureVector forward and reverse overlap at each end. Each gene will be separated with a T2A self-cleaving peptide sequence in order to improve upon the overall lycopene biosynthesis of the existing BioBrick part X which relies on multiple ribosome binding sequences within the DNA sequence. This sequence will then be used in conjunction with the SureVector expression vector assembly kit to produce two plasmids that can be used express the three genes in either E.coli or S. cerevisiae. The E.coli expression vector will contain a pUC replication origin as well as an Ampicillin resistance gene for selection of successful transformants. A T7-promoter will be used to regulate transcription of the CrtE, CrtB and CrtI genes in E. coli. The S. cerevisiae expression vector will contain a yeast autonomous replication sequence (yARS) as well as a LEU2 auxotrophic marker to allow for expression in Leucine deficient strains of S. cerevisiae. A Leucine knockout wildtype S. cerevisiae brewing strain will be created through the use of two BioBrick parts LEU2-L and LEU2-R that we have had synthesized. These parts are designed to knockout the LEU2 gene in S. cerevisiae through the process of homologous recombination. Any gene of interest inserted between these two parts will therefore replace the LEU2 gene within S. cerevisiae creating an auxotrophic strain that can be selected via supplementation of the growth media with leucine. For the purposes of this experiment we will be inserting a KanMX resistance gene that provides resistance to the antibiotic Geneticin (G418). Once these three parts have been assembled into a single plasmid we will PCR amplify the sequence to create a linear sequence that can be used to transform a wildtype brewing S. cerevisiae strain which will subsequently become auxotrophic for the amino acid leucine. The strains can then be transformed with the Miraculin and Lycopene S. cerevisiae expression vectors containing the LEU2 selection marker. To test whether paired-termini antisense RNA interference can be used to regulate gene expression a partial mRFP knockdown BioBrick will be synthesized and compared to the existing mRFP knockdown BioBrick created by Hokkaido Univeristy iGEM 2014 team. This part will contain a number of mismatched bases in the hairpin structure in an effort to reduce overall stability of the RNAi molecule and subsequently reduce its ability to knockdown the mRFP transcript. The RBS+mRFP (BBa_K516032) and RBS+mRFP RNA interference (BBa_K1524104) BioBricks created by the Hokkaido University 2014 iGEM team will be ligated to a constitutive promoter (BBa_J23103) found within the 2015 distribution kit. Our partial knockdown part will be ligated with the same promoter and each knockdown will be co-transformed with the mRFP expressing plasmid. RFP floresence will then be measured to determine whether the partial knockdown part we created reduces RFP expression when compared to the full knockdown part.Protocols
SLiCE/Ex vivo DNA Assembly i.e. Cheap Gibson
Filtration method for competent cells preparation
Preparing liquid YEPD media
Preparing YEPD agar plates
Electroporation Transformation of E. coli DH5aplha
Electroporation Transformation of S. cerevisiae
Experiments
Expressing Miraculin in S. cerevisiae
View Results
Expressing Lycopene in E.coli and S. cerevisiae
View Results
Creating Leucine auxotrophic S. cerevisiae strains
View Results
RNA interference based regulation of RFP in E. coli
View Results
