pRha + CsgA with Hydroxyapatite-affinity tag & pTet + RFP
The DNA was synthesized. The sequence originates from the genomic DNA of E. coli K-12 MG1655 (http://www.ncbi.nlm.nih.gov/gene/949055).
Upon induction with rhamnose, a fusion protein of CsgA with a hydroxyapatite affinity tag is expressed. The CsgA protein monomers can aggregate to form amyloid nanofibres. The affinity tag is supposed to increase adhesive properties of the biofilm created as a result of the CsgA induction. In order to better visualize the individual cells in the biofilm, we added the constitutive expression of RFP to our plasmid in a different operon. For our design, we took advantage of the double terminator present in the biobrick I13521 by placing it in front of our genes.
For our design, we took advantage of the double terminator present in the biobrick I13521 by placing it in front of our genes.
pRha + CsgA + Hydroxyapatite-affinity tag
This part was synthesized. The rhamnose promoter was used by the iGEM 2014 TU Delft team and originally added by the iGEM12 Paris Bettencourt team. RBS and CsgA originate from E. coli K-12 MG1655. The nucleotides encoding for the second and third amino acid of the CsgA gene were changed to optimize synthesis success. (silent mutations). Upon induction with rhamnose, CsgA is expressed and exported into the extracellular space. There, the membrane protein CsgB can trigger the CsgA protein monomers to aggregate and form amyloid nanofibres.
The stop codon needed to be removed from CsgA to add the N-terminal hydroxyapatite affinity tag.
Stefan Robert Marsden, Hector Sanguesa Ferrer
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
**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.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
This scar is part of a synthesized construct.
Scar in constructs BBa_K1583100 - BBa_K1583112
This scar originates from the part BBa_K1316015, on which the composite part BBa_K1583100 is based.
Amplification of the plasmid pJOE3075 (Dr. Altenbuchner).
L-rhamnose-inducible promoter is capable of high-level recombinant protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose by the addition of D-glucose.
One base pair modified (90: A -> T) to avoid EcoRI site. Mutation made in a less conserved base pair.
CsgA missing the stop codon for creation of fusion proteins
This part was designed to create a fusion protein of CsgA with Mfp3 (mussel foot protein). This protein has got high adhesive properties towards wet polar surfaces. Like this, an amyloid nanowire with high binding properties is created.
The stop codon was deleted to create a fusion protein.
The sequence was obtained from the paper "Identification of a Highly Specific Hydroxyapatite-binding Peptide using Phage Display" [1] 1. M. Roy, S. Stanley, E. Amis, M. Becker, Adv. Mater., 2008, 20, 1830-1836.
This part generates a peptide tag which shows high adhesive properties towards hydroxyapatite, a main component of teeth.
No specific design considerations were made.
The DNA was synthesized. The sequence of CsgA originates from the genomic DNA of E. coli K-12 MG1655.(http://www.ncbi.nlm.nih.gov/gene/949055). The design was based on the paper "Programmable biofilm-based materials from engineered curli nanofibres"[1] 1. Nguyen, P. Q., Botyanszki, Z., Tay, P. K. R., & Joshi, N. S. (2014). Programmable biofilm-based materials from engineered curli nanofibres. Nature communications, 5.
A linker with the protein sequence GSGGSG was placed in between the CsgA and the affinity tag for increased performance reasons. The design was based on the paper "Programmable biofilm-based materials from engineered curli nanofibres"[1] 1. Nguyen, P. Q., Botyanszki, Z., Tay, P. K. R., & Joshi, N. S. (2014). Programmable biofilm-based materials from engineered curli nanofibres. Nature communications, 5.
We followed the design of "Programmable biofilm-based materials from engineered curli nanofibres"[1] 1. Nguyen, P. Q., Botyanszki, Z., Tay, P. K. R., & Joshi, N. S. (2014). Programmable biofilm-based materials from engineered curli nanofibres. Nature communications, 5.
Conversion of the iGEM parts registry to SBOL2.1