Difference between revisions of "Team:Austin UTexas/Parts"

Registry Parts

Basic Parts

We submitted two basic PhytoBrick parts and one basic BioBrick part to the registry. Each part is described below. The link to each page is provided in bold text.

Constitutive P8 and P32 Lactococcus lactis promoter and RBS basic parts

The P8 and P32 constitutive promoters and RBS sequences are natively found Lactococcus lactis. Although the transcriptional efficiency of these promoters has been characterized and tested in Lactococcus lactis and other Gram-positive bacteria, their functionality in Gram-negative species such as E. coli has not been recorded in the literature. Thus, we assessed the functionality of these promoters in E. coli using the E-2 Crimson reporter gene, which encodes a red fluorescent protein. We have confirmed that E. coli transformed with cassette plasmids containing these P8 and P32 reporter gene constructs are able to successfully express the E2-Crimson red fluorescent protein. As a PhytoBrick compatible part containing the proper overhangs, the P8 promoter can be assembled into a transcriptional unit via the Golden Gate assembly method to upregulate expression of a gene of interest in E. coli as well as L. lactis.

The constitutive P8 promoter and RBS PhytoBrick basic part can be found on the iGEM registry as: BBa_K2253000. The constitutive P32 promoter and RBS PhytoBrick basic part can be found on the iGEM registry as: BBa_K2253001.

Codon-optimized Blue Chromoprotein basic part

Chromoproteins are non-fluorescent reporters that indicate a change in a part or device through the appearance of coloration. The blue chromoprotein,BBa_K592009, is genetically unstable due to a high metabolic burden caused by its sequence. Our goal was to attempt to reduce this burden through codon optimization, which uses varying set of codons in proportions that are ideal for the specific organism the gene of interest is placed within. Multiple codons can code for the same amino acid but some combinations are used more frequently than others due to an organism’s preference.

In order to stabilize the blue chromoprotein to engineer a plasmid that is capable of maintaining its function that will show and maintain its color for effective long-term use as a reporter, we first ordered a codon-optimized G-block of the original BBa_K592009 chromoprotein. This sequence was made BioBrick compatible via BioBrick assembly and later inserted into the K608002 vector with the pSB1C3 backbone. This procedure was done by digesting both the codon optimized blue chromoprotein and the K608002 vector, ligating the blue chromoprotein insert to the K608002 vector and then conducting a transformation via electroporation. The transformation was then miniprepped and sequence verified.

The codon optimized blue chromoprotein basic part can be found on the iGEM registry as: BBa_K2253002.