We submitted two basic PhytoBrick parts and one basic BioBrick part to the registry. Each part is described below.
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. These promoters were central to our project, as they were used to direct overexpression of the gadB gene. Although the transcriptional efficiency of these promoters has been characterized and tested in Lactococcus lactis and other Gram-positive bacteria such as Lactobacillus, their functionality in Gram-negative species such as E. coli has not been recorded in the literature. Since we wanted to use E. coli as a chassis for creating our various Golden Gate plasmids, we sought to characterize the functionality of these promoters in E. coli through the use of the E2-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 PhytoBrick compatible parts containing the proper overhangs, the P8 and P32 promoters can be assembled into transcriptional units via the Golden Gate assembly method to upregulate expression of genes of interest in E. coli as well as L. lactis and Lactobacillus species. These PhytoBrick promoter parts have been sequence verified.
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 metabolic load through codon optimization, which favors the use of codons that are in proportions 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 for long-term use as an effective color reporter, we first ordered a codon-optimized gBlock of the original BBa_K592009 chromoprotein. This sequence was made BioBrick compatible via BioBrick assembly and later inserted into the BBa_K608002 vector containing a strong promoter and RBS set within the pSB1C3 backbone. This procedure was done by digesting both the codon-optimized blue chromoprotein and the BBa_K608002 vector, ligating the blue chromoprotein insert to the BBa_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.