Composite carrying the Lac-inducible CDI-operon, Lac-inducible RFP and LacI
The CDI-operon was PCR amplified from genomic DNA of E.coli strain EC93. Other parts were taken from the registry.
The composite part contains a lac-inducible CDI operon which gives your strain the capability to inhibit growth of bacterial cells that express BamA in their outer membrane (most gram negative bacteria). Additionally the part contains mRFP1 (BBa_E1010) driven by another lac-inducible promoter as induction control. Moreover the composite part contains LacI. IPTG-induced cells harboring BBa_K1650007 express CdiB which forms a beta-barrel structure. The CdiA protein is transported through CdiB and is displayed on the cell surface. When getting in contact with BamA of a target cell, the C-terminal domain is delivered into the target cell acting as pore forming protein thus destroying the proton motoric force of the target cell. BBa_K1650007 could be used to improve transformation efficiency. Induced CDI+ cells would kill false positive cells.
BBa_K1650007 was toxic for cells in pSB1C3 backbone, presumably because of the high metabolic burden. In accordance with iGEM Headquarter, we submitted BBa_K1650007 in the low copy pSB4C5 backbone
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.
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.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
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 this regulator. This part is incompatible with environments containing lactose or lactose analogs.
**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.
represillator of Elowitz and Leibler (2000)
Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator <bb_part>BBa_R0010</bb_part> and PLlac01 hybrid regulator <bb_part>BBa_R0011</bb_part> and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degredation tail (LVA) has been added to improve the High to Low performance of this part.
References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] Sequence taken from the repressilator of Elowitz and Leibler (2000). The obtained sequence was compared to the wild-type sequence for LacI obtained through a database search. The sequence had been modified from the wild-type in that wild-type GTG start was changed to an ATG start (note, actual ORF in E.coli has several GTG starts it would seem). The LVA tag has been added for quicker degradation. Incompatible with systems containing LacI, lactose, or IPTG.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
Anderson promoter J23106 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Terminator to be placed downstream of multiple cloning site in new pSB series plasmids. Similar to BBa_B0051.
identical to E. coli MG1655 between tonB and yciA
Constitutive Promoter from promoter family of the Berkeley Team 2006 expressing GFP.
Sequence verifying before using to ensure correct sequence in order to use advanced cloning techniques.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1