pRha + Mfp5_CsgA_His fusion protein & 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).
The fusion protein of Mfp5 with CsgA and N-terminal His tag was improved by insertion of constitutively expressed RFP in a different operon to make the cells red fluorescent.
The RFP operon was placed in front of the coding operon to take advantage of its double terminators.
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 the article "Strong underwater adhesives made by self-assembling multi-protein nanofibres". 1. C.Zhong, T.Gurry, A.Cheng, J.Downey, Z.Deng, C. Stultz, T.Lu, Nature Nanotechnology, 2014, 9, 858-866.
No special design considerations were made.
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.
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.
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.
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
Conversion of the iGEM parts registry to SBOL2.1