Synthetic, Phage T7, Phage Lambda, Pseudomonas mendocina.
To tightly repress the nuclease genes (T7 gene 6, PmeI) an antisense promoter and the cI repressor are used. The promoter for expression of the integrase plasmid (the sense promoter) is not on this part. However, the design calls for the Lambda Pr promoter. The part is designed to selectively degrade DNA in vivo.
The main design consideration was to have maximal repression of the toxic nuclease genes.
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
Promoter (lacI regulated, lambda pL hybrid)
represillator of Elowitz and Leibler (2000)
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for strong promotion that can nevertheless be tightly repressed by LacI, the Lac inhibitor (i.e. repressor) (<bb_part>BBa_C0010</bb_part>) ([LUTZ97]). The activity of the promoter can be regulated over a >600-fold range by IPTG in E.Coli DH5-alpha-Z1 (same paper reference).
hybrid promoter design to create strong promoter that is, at the same time, highly repressible. note that the upstream operator installed in this hybrid is slightly different than the one in the original source (Lutz and Bujard, 1997). the most upstream operator region is slightly truncated in the represillator version, so that both operators in the hybrid are the same sequence. see references for details. also, the sequence has been truncated after the transcriptional start site. LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and increase transcription. This part is incompatible with environments containing lactose or lactose analogs.
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
This is the T7 gene 6 ORF, that codes for the T7 gene 6 5'-3' exonuclease. For properties of T7 gene 6 exonuclease see: Kerr and Sadowski "Gene 6 Exonuclease of Bacteriophage T7" The Journal of Biological Chemistry Vol. 247 No. 1 January 10, pp 311-318, 1972.
We needed to change the stop codon to the standard stop codon TAA. We later realized that we should have added tandem TAA. The Tandem TAA stop codon is present in the composite parts containing this ORF. The ORF alone has the single TAA stop codon.
Convergent Promoter System: Reverse Module: rPlac+TT
This part contains a reverse BBa_R0011 followed by a forward TT, BBa_B0015.
The forward and reverse efficiencies of TTs in the registry had to be considered, especially for this part's convergent partner, BBa_K093003. R0011 was used instead of R0010 to avoid catabolite repression. The idea is to place BBa_K093003 upstream of the gene of interest, that requires tight repression, and to place BBa_K093004 downstream. If the cells are grown in the presence of IPTG POlacI is induced and anti-sense mRNA is produced. Anti-sense mRNA will repress expression of the gene of interest through RNAi. This system also requires cI repressor (BBa_C0051) under control of POlacI (BBa_R0011). This system would be functional in a laqIq strain such as E. coli. TG1. O'Connor,C.D., & Timmins, K.N. (1987). Highly repressible expression system for cloning genes that specify potentialy toxic proteins. Journal of Bacteriology. 169, 4457-4462.
cI repressor, BBa_C0051, with Elowitz RBS, BBa_B0034.
We wanted to express PmeI. That requires a RBS.
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
This is the PmeI ORF, coding for the PmeI restriction endonuclease gene with the recognition sequence: GTTTAAAC
Conversion of the iGEM parts registry to SBOL2.1