Difference between revisions of "Team:London Biohackspace/experiments/rna-based-regulation"

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                         <h4>Introduction</h4>
 
                         <h4>Introduction</h4>
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                             <p>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.</p>
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                             <p>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.</p>
 
                         <h4>Materials and methods</h4>
 
                         <h4>Materials and methods</h4>
 
                         <h4>Results</h4>
 
                         <h4>Results</h4>

Latest revision as of 23:24, 18 September 2015

BEER.CO - Royal quality

  • Lab Book

Experiments

RNA INTERFERENCE BASED REGULATION OF RFP IN E. COLI

Introduction

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.

Materials and methods

Results

Discussion

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