LuxR/AHL driven Lysis Cassette with Protein level Timer Delay
The quorum sensing components come from Vibrio fischeri. The cI components hail from the T4 enterobacteria phage.
This system presents an AHL/LuxR dependent mechanism for delayed cell lysis. Following induction with AHL, the repressible pLux becomes inhibited and ceases producing repressor protein cI. Following this the pcI becomes uninhibited and proceeds to translate regions encoding lysis inducing proteins. Taken together with the need for AHL/LuxR complexation and cI's ceased production, this exemplifies a time delayed quorum sensing dependent kill switch for the cell. Note: this part does not include the LuxR coding region. Without this within the plasmid as well, the system will not function.
We had to balance potential basal levels of lysis expression. Luckily, this proved not to be an issue.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
cI repressor from E. coli phage lambda (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator (BBa_R0051).
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 BBa_C0051 cI repressor is based on the cI repressor from the Elowitz's repressilator. It has been modified to include a rapid degradation LAA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Incompatible with host expressing cI repressor.
This part involves the -10 binding site, the -35 binding site, and the twenty nucleotides between that constitute the lux box. With this part, LuxR functions as a acyl-homoserine lactone-dependent repressor. LuxR resonds to the HSL produced by LuxI, N-(3-oxohexanoyl)-HSL. The Lux box is positioned such that it partially overlaps the consensus -35 and -10 hexamers of an RNA polymerase binding site.
Srini Devadas, David Gray, Ronny Krashinsky, Debra Lin, and Chris Zheng Liu
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