R Protein (Endolysin: Phage lambda lysozyme)
S' Protein (Holin) / S Protein (Anti-holin)
Lysis protein S from Enterobacteria phage lambda (codon usage optimized for R. opacus)
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.
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.
T4 endolysin under the control of T7 promotor
Ane Fernanda Beraldi Zeidler, Luige A. Llerena Calder??n and Marcos Henrique de Moraes
This construction is a device composed of: BBa_I746911 - BBa_K112806 - Bba_B0014. This device contain the T4 endolysin, a lysozyme from enterobacteria phage, that degrades peptidoglycan layer, under the control of the T7 promoter.
For more accurate tests about the endolysin effectiveness, this part has to be under the control of an inducible promoter, like the T7 promoter.
This device is a junction of previously designed parts: BBa_I746911 - Designed by Stefan Milde Group: iGEM07_Cambridge (2008-09-30) BBa_K112806 - Designed by Jin Huh Group: iGEM08_UC_Berkeley (2008-10-31) Bba_B0014 - Designed by Reshma Shetty Group: Registry (2003-07-16)
Bacteriophage Lysis Cassette S105, R, and Rz
The gene cassette consists of the S105 protein (Holin), R protein (Endolysin), and Rz protein. When paired with an inducible promoter, the cassette is activated by the inducer and begins to produce the proteins which lyse the cell. This cassette contains the mutant S gene, which can be found individually as BBa_K124014. The S variation allows for faster lysis.
The DNA for this part was graciously donated to the Brown iGEM Team by Ry Young at Texas A&M University. The team then BioBricked the part for submission to the registry.
Lysis inhibitor from Enterobacteria phage lambda (codon usage optimized for R. opacus)
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.
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.
Lysis gene (promotes lysis in colicin-producing bacteria strain)
This lysis gene encodes for the lysis protein in colicin-producing strains of bacteria. Once activated, it causes the host cell to lyze. It also removes the immunity protein out of colicin, and hence, activates the endonuclease activity of the colicin.
Endolysin is a lysozyme which cleaves the glycosidic bond between the C-1 of N-acetylmuramic acid (NAM) and the C-4 of N-acetylglucosamine (NAG) in the bacterial peptidoglycan (PG). This leads to the complete lysis of the cell. Holin is a protein that assembles together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer. It is sourced from T4 bacteriophage. This composite part can be used to induce self-destruction with a regulatory device in the upstream region.
There is no RBS in the upstream region, so during the assemble procedure, this must be taken care of.
Gene sequences of endolysin and holin are gathered from the Registry.
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.
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.
Receiver cell, T4 lysis device is under pLas.
We got BBa_K772004 from 2012 Fatih Medical team.
LasR Device:K772004 Enterobacteria phage T4 Lysis Device - no promoter: K112808
This part causes cell death. It contains holin/antiholin, endolysin and Rz/Rz1 genes.
We made point mutation and prepared wild type phage DNA.
λ -Hind Ⅲ digest (product of TaKaRa Bio)
Tais Sineiro Herig, Raissa Estrela Curado and Gleidson Silva Teixeira