This part originates from the E.coli K-12 MG1655 genome and from mussels. 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 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. By creating a fusion protein, the adhesive properties of the mussel foot protein is combined with the formation of nanowires. 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).
A mutation had to be introduced at base pair 766 to remove a PstI restriction site. The protein sequence was not changed in doing so.
Non-coding scar site in between RBS and gene
The RBS and CsgC were synthesized from the genomic sequence of E.coli K-12 MG1655. The promoter is part of the constitutive promoter family made by John Anderson from iGEM Berkeley 2006.
CsgC is positively involved in the extracellular aggregation of CsgA into amyloid nanowires (curli assembly) in E.coli functioning as a chaperone for CsgA.
We inserted non-coding scar sequences after the promoter and RBS to prevent missing initial codons during transcription/translation.
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 Mfp5 (mussel foot protein) has high adhesive properties towards wet polar surfaces.
No special considerations were required.
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.
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.
Spacer in between promoter and coding sequence
This sequence was designed as a spacer to prevent errors in transcription by placing the RBS too close to the promoter.
This sequence was designed as a spacer to prevent errors in transcription by placing the RBS too close to the promoter. Formation of a hairpin structure or self complementary sequences was avoided.
Spacer in between coding sequence and suffix
Non-coding and non-self-complementary codons were chosen at random.
Designed as a spacer of 6 base pairs between our coding sequence and the biobrick suffix.
Hairpin formation and self-complementary sequences were avoided.
CsgA missing the start codon for fusion protein
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. An illegal PstI restriction site (766) in CsgA had to be mutated (A->G) not changing the protein sequence.
In order to create a fusion protein of CsgA with Mfp5 in front of it, CsgA was designed without its start codon.
An illegal PstI restriction site (766) in CsgA had to be mutated (A->G) not changing the protein sequence.
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.
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.
Conversion of the iGEM parts registry to SBOL2.1