Curli Fiber generator under the control of T7 promoter with a relatively strong expression of CsgA,C
CsgB and CsgAC were collected by PCR from genomic DNA of E coli. DH5alpha strain. And site-directed mutation by overlap PCR primers. Others parts come from Biobricks Attribution Kit.
This part contains the major subunit of curli fiber CsgA and its nucleator CsgB, in which CsgA is constitutive expression(a relatively strong promoter compared to BBa_K1361001) whereas CsgB is under the control of T7 promotor. This part cannot functioning alone without expression of CsgEFG genes in the same cell, for they coding the outer membrane channel secrete system for CsgA and CsgB.
The PstI loci within CsgA sequence has been replaced from CTGCAG to CTGCCG.
Double terminator consisting of BBa_B0010 and BBa_B0012
Strong promoter, strong RBS combination for high expression levels of proteins
It is a combination of strong promoter (J23100) and RBS (B0034).
-J23100+B0034 -Strong promoter, strong RBS combination for high expression levels of proteins Consensus constitutive promoter and RBS sequence-produces strongest possible expression.
Josh Atkinson, Mike Ferguson, and Ben Parker
Curli Fiber assembly associated protein CsgA, CsgC
PCR on genomic DNA from E coli. DH5alpha strain. And site-directed mutation by overlap PCR primers.
CsgA, CsgC genes are directly PCRed from E coli. DH5alpha genome as a whole. There is a scar sequence between CsgA and CsgC coding region that contains a promoter sequence may constitutively transcript CsgC gene. CsgA protein is the major subunit of Curli Fiber. CsgC may regulate CsgG outer membrane assembly of channel protein CsgG and pore activity through modification of C230 in CsgG.
CsgB, curlin nucleator protein, minor subunit in curli complex
PCR on genomic DNA from E coli. DH5alpha strain.
CsgB is a secretive protein that attached cells' outer membranes. It plays key role in curli fiber polymerization. CsgB is the nucleator for CsgA aggregation in vivo, that facilitate the transformation of soluble CsgA into polymer fiber.
CsgB can be designed under inductively control while make CsgACEFG genes constitutively express respectly. Using this method could achieve the immediate synthesis of curli fiber after inducer has been added.
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.
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.
Conversion of the iGEM parts registry to SBOL2.1