Curli Fiber generator under Pbad induction with positive regulation
CsgAC,CsgB collected by PCR from genome of E coli DH5alpha respectively. Other genes are collected from Biobricks Attribution Kit.
We assembled this part in order to examine the concept that change of cell motility by control of CheZ chemotaxis regulator could disturb pre-expressed curli fiber structure. If the inducer occurs, in this case_acacia, CsgB protein will secrete into extracellular and act as nucleator to recruit soluble CsgA monomer into amyloid fibres, and the transcription of CheZ will be downgrade via cI-Plamda NOT gate. If induction condition disappears at a certain point, suppression of CheZ will be discharged. As the result, cells will be more moveable thus disturb or inhibit the establishment of curli structure. And in our final device, the modified CsgA that has ability to absorb nanogold, thus the dynamic detection could achieve by monitoring current change between two electrodes in the culture.
The PstI loci within CsgA sequence has been replaced from CTGCAG to CTGCCG. And for nano gold absorbability, 6xLinker+2xCys(GGCAGCGGTGGCAGCGGTGGCGGT TGTTGC) and LPFFD(CTGCCGTTCTTTGAT) were added at the C-treminal of CsgA protein respectively. CsgA with7xhis tag at C-treminal were also used for Au-NTA-Ni absorbing.
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.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Incompatible with host expressing cI repressor.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
Double terminator consisting of BBa_B0010 and BBa_B0012
cheZ, chemotaxis regulator, protein phosphatase for CheY
Polar localization is dependent on CheA-short. CheZ is a cytosolic phosphatase which functions in the chemotaxis signal transduction complex. Plays an important role in bacterial chemotaxis signal transduction pathway by accelerating the dephosphorylation of phosphorylated CheY (CheY-P).
how to control the proper expression of cheZ
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.
The PstI loci within CsgA sequence has been replaced from CTGCAG to CTGCCG.
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.
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.
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.
Conversion of the iGEM parts registry to SBOL2.1