Difference between revisions of "Part:BBa K2282006"

Revision as of 12:20, 30 October 2017

AmilCP with DSbox (BBa_K2282001) supplemented with BBa_J23100, BBa_B0034 and a double terminator.

Usage and Biology

The DSBox (Downstream Box) increases the translation of the mRNA at low temperature, probably by binding to the 30s subunit via ribosomal protein S1 (Studer et al 2006, Duval et al, 2013, Qu et al 2012). The DSBox is unusual among 5' regulatory sequence motifs in that it appears after the start codon and is translated into the protein product at the N-terminus. The double terminator provides robust termination. AmilCP is a coral chromoprotein which displays a strong blue color. With this part we can evaluate the impact of the DSBox on the coding sequence of the protein and see if amilCP expression is modified as well as its properties (colors, time of folding).

Source of this part

This part is the same as BBa_K2282005 with the slight difference that we prepended the DSBox sequence (Mitta et al, 1997) from the E.coli genome to the amilCP coding sequence. The DSBox size as defined in Mitta et al, 1997 is 14bp. In order to avoid introducing a frame shift error we added an adenine nucleotide at the 3’ end to increase the size from 14bp to 15bp by completing the 3’ codon. The sequences of the DSBox and other cold-induced translational regulatory elements are very similar to the parts of the iGEM10_Mexico-UNAM-CINVESTAV that can be found here : http://partsregistry.org/Part:BBa_K328003

Design consideration

Adding 15 nucleotides to a protein coding sequence raised some concern about its effect on protein stability and activity. We addressed this problem with molecular modelling to optimize the placement of the DS Box (see our wiki) and confirmed the results in the lab.

Sequence and Features