The holin used in the kill gene comes from a bacteriophage.
This part contains a kill gene that uses holin to produce holes in the cell wall and destroy the cell if the cell is below 42 degrees Celsius or in the presence of tetracycline. RFP is used as a reporter to visually alert the researcher to a cell which is marked for death.
This gene was designed to allow for expression of the reporter gene previous to the expression of the kill gene.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Double terminator consisting of BBa_B0010 and BBa_B0012
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=160221&to=160877&view=gbwithparts
Holins from T4 bacteriophage assemble together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer.
Making composite part of rbs.holin is problematic unless antiholin is expressed in bacteria together. Antiholin binds holin and inhibits the formation of holin.
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=66503&to=66997&view=gbwithparts
The lysozyme from enterobacteria phage T4 degrades peptidoglycan layer.
High expression of lysozyme only is toxic to bacteria.
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=59202&to=59495&view=gbwithparts
Antiholin binds holin and prevents holin multimers from forming pores on the inner membrane of bacteria.
Higher constitutive expression of antiholin compared to the expression of holin is required for the stable lysis device.
Transcriptional terminator consisting of a 64 bp stem-loop.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Conversion of the iGEM parts registry to SBOL2.1