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Biobricks designed

BBa_K836006

Lysis protein S from Enterobacteria phage lambda. This gene codifies for a protein that makes holes in the inner membrane of the bacterial cell. Between the RBS and the CDS a spacer consisting on a scar from a standard suffix and a prefix for CDS was inserted.

Source: http://www.uniprot.org/uniprot/P03705#section_seq it was optimized for R. opacus (avoiding standard restriction sites)

Part:BBa_K836005

Lysozyme from Rhodococcus phage RER2. Protein involved in the peptidoglycan degradation of the bacterial cell wall. A RBS for Gram positive bacteria (SpoVG) was added at the beginning.A spacer was put between the RBS and the start codon which corresponds to the scar between a normal suffix and the prefix used for CDS to avoid RBS-CDS problems. The sequence has no termination codon since we rely on the double terminators of the scar formed during assembly of biobricks.

Source: http://www.uniprot.org/uniprot/G9FHW6) and then it was optimized for R. opacus (avoiding standard restriction sites).

Part:BBa_K836007

Nitrilase regulator from R. rhodochrous. Protein in charge to regulate the expression of the genes put under control of nitA promoter. A spacer which consist of the scar left by the assembly of a normal suffix and a CDS prefix was inserted between RBS and CDS. The sequence has no termination codon since we rely on the double terminators of the scar formed during assembly of biobricks.

Source: The amino sequence was obtained from Uniprot (http://www.uniprot.org/uniprot/P72312) and then the condon usage was optimized for expression in R. opacus.

Part:BBa_K836008

nitA promoter. Promoter inducible by nitrile and similar agents.

Source: http://www.ncbi.nlm.nih.gov/nucleotide/346420896?report=genbank&log$=nuclalign&blast_rank=1&RID=W0SH1PA7016

Part:BBa_K836009

Lysis device for R. opacus. Lysis construct based on the one presented by Berkeley's 2008 team BBa_K112022. It is based on the inducible expression of a lysozyme and lambda phage's holin in order to liberate internal products of the bacterial cell in an efficient way. The induction is achieved by the addition of nitrile or a similar agent since both genes are regulated by nitA promoter. Constitutive expression of nitR and a low constitutive expression of an antiholin are required to avoid undesired lysis.

Source: The majority of the parts where made by backtranslating a protein. Others are existing biobricks while some are promoters obtained from databases as NCBI.

Part:BBa_K836002

O-acyltransferase WSD from Acinetobacter sp. This biobrick codifies for the protein DGAT (Diacylglycerol O-acyltransferase) which have dual activity, it can act as a wax ester synthase or as a diacylglycerol acyltransferase. In the latter case, its function consists in mediate the final step in TGAs synthesis using a fatty acyl-CoA and a DGA. This enzyme is vital in the storage and synthesis of lipids. Two transcriptional terminators (two biobricks BBa_B1006) were set at the end of the coding sequence.

Source: The amino sequence of this part was taken from Uniprot (wax-dgaT accesion Q8GGG1; http://www.uniprot.org/uniprot/Q8GGG1) and then codon optimization for R. opacus (avoiding restriction sites for the standard assembly method)

Part:BBa_K836003

lipA from B. cepacia. This biobrick codifies for the protein lipA from Burkholderia cepacia (Pseudomonas cepacia) which catalyzes the hydrolysis of triglycerides (Triacylglycerol + H2O = diacylglycerol + a carboxylate). In the presence of certain alcohols, it catalyzes the transesterification of TGAs to produce acyl esters and glycerol. It has only one PTM (Post-Translational Modification) which consists in a sulfide bond between 234 and 314 aa. Calcium serves as a cofactor for this enzyme (1 ion per subunit). The native signal peptide was not modified. The sequence of a transcriptional terminator (biobrick BBa_B1006) was set at the end of the coding sequence.

Source: The amino sequence of this part was taken from Uniprot (lipA accesion P22088; http://www.uniprot.org/uniprot/P22088) and then codon optimization for E. coli (avoiding restriction sites for the standard assembly method).

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