Pgad: PCR from vector from CUHK 2011 team Lysis: registry
This construct was developed by Northwestern's 2012 iGEM team as part of a phytase producing vector meant to self lyse in high chloride and low pH conditions. The device is designed to lyse in the presence of high intracellular chloride concentrations.
Pgad - Intracellular Chloride-Sensing Cassette
The gene sequence was obtained from the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes PgadR, gadR, Pgad and the starting codon ATG.
Pgad is chloride-sensitive promoter which was first discovered in Lactococcus lactis1, which is a gram-positive bacterium which can live in acidic environment. In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of Pgad2. The gene before Pgad, named gadR, which is constitutively expressed under the control of PgadR, is a positive regulator of Pgad coupled genes while intracellular chloride is level elevated.
Synthesized part by GenScript,all the illegal sites for RFC 10 (EcoRI, XbaI, SpeI, PstI) have been substituted by other codon producing the same amino acid in E. coli.
Enterobacteria phage T4 Lysis Device - no promoter
Pubmed Accession Number: NC_000866 Enterobacteria phage T4
This is the lysis device using three genes from enterobacteria phage T4: lysozyme, holin, and antiholin. Various promoters can be installed with this device to control the lysis of bacteria. Antiholin has its own constitutive promoter to prevent the formation of holin multimers from basal expression. When the device is off, higher expression of antiholin compared to holin is required for better stability of the device.
[Pcon].[Antiholin].[terminator] was assembled first, and then assembled with [T4 holin].[terminator]; Constitutive expression of antiholin is required before assembling holin.
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
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.
Conversion of the iGEM parts registry to SBOL2.1