Curli Fiber generator where CsgBtrunc, a dissociative nucleator, under the control of Pbad promoter
CsgAC and CsgBtrunc were collected by PCR from genome of E coli DH5alpha respectively.
This part contains the major subunit of curli fiber CsgA and its nucleator CsgBtrunc, in which CsgA is constitutive expression whereas CsgBtrunc is under the control of Pbad promotor. CsgBtrunc is different from native CsgB from the deletion of C-terminal amino acid from 133 to 155 that it cannot attach cell outer membrane. And CsgBtrunc itself is highly aggregative in the culture. This part is designed to be used for immediate generate of large amount of curli fiber after adding of the inducer and a stronger promoter for CsgA was chosen to parallel with BBa_K1361002. 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.
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
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.
CsgBtrunc, curli fiber nucleator with a C-terminnal deletion
This part is collected by PCR from genome of E coli. DH5alpha.
This part is Curli fiber nucleator subunit protein CsgB with deletion of C-terminal amino acid from 133 to 155 that is lost the ability to attach outer membrane of the cell.
Deletion is designed referring Hammer, Neal D., Jens C. Schmidt, and Matthew R. Chapman. "The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization." Proceedings of the National Academy of Sciences 104.30 (2007): 12494-12499.
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type (<partinfo>I13453</partinfo>) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
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.
Conversion of the iGEM parts registry to SBOL2.1