Curli Fiber generator under the control of Pbad promoter with CsgA modified by His tag
CsgB and CsgC were collected by PCR from genomic DNA of E coli. DH5alpha strain. CsgA-his was synthesized by GeneScrip.
This part contains the major subunit of curli fiber CsgA-his and its nucleator CsgB, in which CsgA-his is constitutive expression whereas CsgB is under the control of Pbad promotor. This part is designed to be used for immediate generate of large amount of curli fiber after adding of the inducer, and also can be used for purification of CsgA by Ni-NTA reign, or absorbing Au-NTA-Ni nano partical. 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 modifying of CsgA was referring the supplementary of Lu, Michelle Y., et al. "Synthesis and patterning of tunable multiscale materials with engineered cells." (2014).
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
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.
Curli Fiber major monomer-CsgA with his tag
This part is CsgA(BBa_K1361999) with twin 7Xhis tag. And can be used for purified of CsgA monomer or absorbing Au-NTA-Ni nano partical. CsgA is the major monomer of curli fiber. In vivo, its alone has little polymerizability and CsgB protein is requested for quick polymerization of CsgA.
CsgC, curli fiber secrete accessory molecules
CsgC were collected by PCR from genomic DNA of E coli. DH5alpha strain.
Within the periplasm, CsgC may regulate CsgG outer membrane assembly and pore activity through modification of C230 in CsgG. And its essential for curli fiber assembly.
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.
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1