Team:Cambridge/Parts

Parts designed and constructed

1.Fluoride Sensitive Riboswitch (Part:BBa_K911003)

Riboswitch that is highly sensitive to the F- ion. We have characterized this part in three different chassis: TOP10 e.coli, Laboratory strain bacillus subtilis and a strain of bacillus subtilis with its normal fluoride riboswitch knocked out (kindly provided by the Breaker lab in Yale). Results of Miller assays for these three chassis are also provided.

2.Synthesised Ratiometric Luciferase construct in non-standard plasmid (Part:BBa_K911004)

This part was designed as a ratiometric luciferase reporter. The first promoter, hyperSpank, is LacI - repressed and controls the transcription of a (vibrio harveyi) luxA gene that has been fused at the N-terminus to an mOrange gene via a flexible linker. This was described by Dachuan Ke and Shiao-Chun Tu (2011) as having an additional peak in its emission spectrum at 560 nm, whereas the normal peak is at 490 nm. This is terminated by b0015. Downstream, pVEG controls the translation of the entire normal lux operon, which is again terminated by b0015. The idea is that the normal luciferase output acts as an internal control signal, to which the output of the induced luciferase with the spectral shift can be normalised. We designed this to be compatible with our cheap open-source sensing hardware - see our wiki for more details. This contruct has been designed to work in bacillus and E.coli, so all the RBSes are at the bacillus consensus. Users looking for a lux operon optimised for B.subtilus may wish to use the one included within this construct. There are some issues with the stability and function of this part necessitating its submission to the registry in a non-standard backbone (BBa_k911006), see experience for further details and characterisation. We contacted the registry about this and were granted exemption from the pSB1C3 standard for this part.

<groupparts>iGEM012 Cambridge</groupparts>