Part:BBa_K1994022:Experience

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Applications of BBa_K1994022

This part was characterised by the Warwick iGEM 2016 team.

Composite was assembled and transformed into DH5alpha E. coli cells. Three versions of this construct were tested using a growth and fluorescence assay, measuring OD600, and emission of GFP at 530nm. This data demonstrates the difference in fluorescence expression between constructs containing the weak omega promoter and a PheA terminator, those containing only the terminator, and constructs containing neither.

The graphs show that expression of GFP is much higher in cells containing the weak omega promoter. There is also negligible difference in growth between cells containing the plasmid with the promoter and without.

This part was also triple transformed into DH5alpha cells also containing pSB3T5-PP7-omega (BBa_K1994024) and a library of various sgRNAs containing a PP7 handle (BBa_K1994021) with 20 different targeting regions cloned into the golden gate site of this part. This was to demonstrate that the library of sgRNAs would increase the expression of GFP by using dCas9 binding to bring an omega factor closer to the weakened omega promoter. However the data collected shows negligible difference in GFP expression between cells containing the library sgRNAs and the negative control that contains all three plasmids, but without an inserted targeting region.

The white diamonds show the negative control with error bars representing a single standard deviation from the mean. The majority of the library sgRNAs fall within the error bars. The difference in expression is not sufficiently different to suggest a correlation between specific sgRNAs and GFP expression compared to the negative control.

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