The majority of the parts where made by backtranslating a protein. Others are existing biobricks while some are promoters obtained from databases as NCBI. Check each individual part to know its origin.
Lysis construct based on the one presented by Berkeley's 2008 team BBa_K112022 (http://partsregistry.org/wiki/index.php?title=Part: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.
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs
Artificial terminator, estimated %T~>90% *8bp stem, 6nt loop *Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 9 residues.
Promoter veg a constitutive promoter for B. subtilis
The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database#3 then a section containing the binding site synthesised by Geneart.
Pveg is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. Pveg contains binding sites for the ''B.sutbilis'' major sigma factor#1. Pveg in ''B.subtilis'' utilises two binding sites to cause high expression of genes#2, however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how#3. The context with which we used the promoter Pveg is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include a single binding site for the ''B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.
Promoter taken from NCBI (http://www.ncbi.nlm.nih.gov/nucleotide/346420896?report=genbank&log$=nuclalign&blast_rank=1&RID=W0SH1PA7016).
Promoter inducible by nitrile and similar agents.
Lysis inhibitor from Enterobacteria phage lambda (codon usage optimized for R. opacus)
The amino sequence was obtained from Uniprot (http://www.uniprot.org/uniprot/P03705#section_seq lysis inhibitor isoform). The codon optimization for R. opacus (avoiding standard restriction sites EcoRI, PstI, XbaI and SpeI) was made using the free software GeneDesigner from DNA 2.0.
This protein is the one in charge to avoid the formation of pores in the inner membrane of the bacterial cell. It has been optimized for expression in R. opacus. A weak RBS for Gram positive bacteria was used (http://partsregistry.org/Part:BBa_K090506:Design)in order to obtain low concentration of the protein.
We wanted a low concentration of this protein since medium to large concentrations could affect or completely inhibit the lysis device. Basal but constitutive expression of this protein is needed to avoid the formation of pores in the design lysis device.
Lysozyme from Rhodococcus phage RER2 (codon usage optimized for R, opacus)
The amino sequence was obtained from Uniprot (http://www.uniprot.org/uniprot/G9FHW6) and then it was optimized for R. opacus (avoiding standard restriction sites).
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
Lysis protein S from Enterobacteria phage lambda (codon usage optimized for R. opacus)
The amino sequence was obtained from Uniprot (http://www.uniprot.org/uniprot/P03705#section_seq lysis protein S) and optimized for R. opacus (avoiding standard restriction sites) using the free software GeneDesigner from DNA 2.0.
This gene codifies for a protein that makes holes in the inner membrane of the bacterial cell. A Gram positive RBS (SpoVG, http://partsregistry.org/wiki/index.php?title=Part:BBa_K143021) was added at the beginning.
Between the RBS and the CDS a spacer consisting on a scar from a standard suffix and a preffix for CDS was inserted.
Nitrilase regulator from R. rhodochrous (codon usage optimized for R. opacus)
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 using the free software GeneDesigner from DNA 2.0.
Protein in charge to regulate the expression of the genes put under control of nitA promoter. A Gram positive RBS (SpoVG http://partsregistry.org/wiki/index.php?title=Part:BBa_K143021) was inserted at the beginning.
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.
Conversion of the iGEM parts registry to SBOL2.1