CRISPR-Cas9 contaiment device repressed by arabinose and tryptophan
This is a prototype containment device utilizing CRISPR-Cas9 system which aims to control accidental leakage of GMM once it escape from medium containing arabinose and tryptophan. The device consists of two modules, firstly a arabinose repressible cas9 expression module and a tryptophan repressible gRNA expression module. Under the presence of sufficient amount of arabinose and tryptophan the device remain silent, however when bacteria escape to environment without arabinose and tryptophan, Cas9 and a gRNA targeting DNA polymerase III alpha subunit will be expressed. Subsequent cleavage of the targeted sequence may hinder DNA replication and
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.
Double terminator consisting of BBa_B0010 and BBa_B0012
Cas9 (optimized for expression in E. coli)
This part code for cas9 originated from S. pyogenes SF 370 with codon optimized for expression in E. coli.
gRNA for cas9 targeting DNA polyerase III alpha subunit
Generated from sequence of DNA polyerase III alpha subunit in E. coli BL21 DE3
gRNA targeting DNA polyerase III alpha subunit in E. coli BL21 DE3
Addition of Trp repress anything downstream of the inverter. Notice that it contain illegal SpeI sites, however it it essential for the functioning of this part.
The two parts are all from previous parts.
A araBAD promoter with araC transcripted from the negative direction.
We generate this regulatory part to regulate our reporter gene expression in our project.
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.
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.
PC and AraC from the AraBAD operator. PC and AraC are on the complementary strand, reading right to left.
Cloned from plasmid pJPR2 described in Paluh and Yanofsky NAR 1986
The sequence contains SpeI sites at positions critical for function. We modified the SpeI site in the biobrick suffix to an NheI site as done by UNAM 2010
Conversion of the iGEM parts registry to SBOL2.1