pRha + CsgA_Mfp3_His fusion protein & pTET + RFP
The sequence originates from the genomic DNA of E. coli K-12 MG1655 (http://www.ncbi.nlm.nih.gov/gene/949055).
The fusion protein of CsgA with Mfp3 and N-terminal His tag was improved by insertion of constitutively expressed RFP in a different operon to make the cells red fluorescent.
I13521 was placed in front of K1583007 to take advantage of the double terminator in I13521.
This DNA was synthesized. The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres".[1] 1. C.Zhong, T.Gurry, A.Cheng, J.Downey, Z.Deng, C. Stultz, T.Lu, Nature Nanotechnology, 2014, 9, 858-866.
Inspired by mussels, the Mfp3 (mussel foot protein) has high adhesive properties towards wet polar surfaces.
No special design considerations were taken.
This amino acid sequence was used as a linker to create a fusion protein from Mfp3/Mfp5 with CsgA.
No special design considerations needed to be taken.
Standard DNA sequence optimized for E.coli.
Addition of a His tag at the N-terminal end of a protein of interest is goal of this basic part.
The codon was optimized for synthesis to avoid repetetive motives of too many codons.
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). The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres".[1] 1. C.Zhong, T.Gurry, A.Cheng, J.Downey, Z.Deng, C. Stultz, T.Lu, Nature Nanotechnology, 2014, 9, 858-866.
This RBS was used in our constructs Mfp5+CsgA and CsgA+Mfp3. CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from E.coli K-12 MG1655. Inspired by mussels, the Mfp5 (mussel foot protein) has high adhesive properties towards wet polar surfaces. This DNA was synthesized. The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres".[1] 1. C.Zhong, T.Gurry, A.Cheng, J.Downey, Z.Deng, C. Stultz, T.Lu, Nature Nanotechnology, 2014, 9, 858-866.
The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres".[1] 1. C.Zhong, T.Gurry, A.Cheng, J.Downey, Z.Deng, C. Stultz, T.Lu, Nature Nanotechnology, 2014, 9, 858-866.
CsgA missing the stop codon for creation of fusion proteins
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).
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.
This scar is part of a synthesized construct. It is used in BBa_K1583007.
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.
This DNA was synthesized. The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres".[1] 1. C.Zhong, T.Gurry, A.Cheng, J.Downey, Z.Deng, C. Stultz, T.Lu, Nature Nanotechnology, 2014, 9, 858-866. The CsgA sequence originates from the genomic DNA of E. coli K-12 MG1655 (http://www.ncbi.nlm.nih.gov/gene/949055).
CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from E.coli K-12 MG1655. Inspired by mussels, the Mfp3 (mussel foot protein) has high adhesive properties towards wet polar surfaces.By creating a fusion protein, the adhesive properties of the mussel foot protein is combined with the formation of bacterial nanowires. The design originates from "Strong underwater adhesives made by self-assembling multi-protein nanofibres" C.Zhong, T.Gurry, A.Cheng, J.Downey, Z.Deng, C. Stultz, T.Lu, Nature Nanotechnology, 2014, 9, 858-866.
A mutation in CsgA had to be introduced to remove a PstI restriction site. This was done without changing the protein sequence and a codon optimizer was used to ensure high expression in E.coli.
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.
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
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.
Conversion of the iGEM parts registry to SBOL2.1