**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.
the source of the gene is from Methylobacterium chloromethanicum. Other parts are from the registry.
the rpsT gene with a promoter-RBS- and two TC stops added
I just found different ribosome binding sites and Tc stops from the registry. The promoter is for the glucose controlled ompC porin gene. The RBS is the native rbs for the sam5 gene of Saccharothrix espanaensis The first stop stops RNA polymerase from transcribing the TetR gene. The second codes for a gene that inhibits cell division
Ampicillin: atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaac gctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaaga tccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcc cgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcac agaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcca acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcctt gatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaac gttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaag ttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtct cgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaac tatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaa
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes the three OmpR binding sites: C1, C2, and C3. The promoter starts at -107 and goes up to the transcriptional start site at +1. An alternate version, BBa_R0083, cuts out the C2 and C3 sites. The efficacy of this promoter versus the truncated ompC upstream region (BBa_R0083) or the ompF upstream region (BBa_R0084) is currently unknown.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
This is the native ribosome binding site for sam5 (4-coumarate-3-hydroxylase) of the bacterium Saccharothrix espanaensis.
This is a virtual part to be added to coding sequence I742141.
rpsT gene from Methylobacterium Chloromethanicum
Methylobacterium Chloromethanicum. The sequence comes from the site http://www.ncbi.nlm.nih.gov/nuccore/218528082?from=359&to=625
The rpsT gene binds directly to the 16s rRNA and induces post translational inhibition of arginine and ornithine decarboxylase
I copied and pasted the sequence from NCBI.
This Construct will allow for the detection and degredation of BPA in cells already containing the ER (K123003) biobrick. It works in two systems first. Together K123003 and K123009 produce a NOT gate where when Bisphenol A (BPA), Estrogen or any other estrogen mimic is in the system the product of K123003 dimerizes and attaches to the ERE site of K123009. This stops the production of TetR (R0040) by phsically stoping the Polymerase from transcribing it. This allows the pTet(C0040)promoter to turn on the production of RFP (E1010) for detection as well as BisdA(K123000) and BisdB(K123001)which allow for the degredation of BPA.
While BisdA and BisdB have only been shown to degrade BPA we have put in RFP to allow for the detection of other estrogen mimics as well.
''Bacillus subtilis'' in response to stress such as DNA damage stops the cells from dividing. This is a part of the SOS response initiated by the accumulation of single stranded DNA from DNA damage or stalled replication. Two proteins are vital for this response: RecA and LexA. RecA forms filaments on ssDNA and promotes the autocleavage of LexA. LexA usually represses the SOS operon. ''dinR'' is homologous to ''lexA'' in ''E. coli'' and is transcribed in the opposite direction of ''yneA''. ''yneA'' stops the formation of ''ftsZ'' ring indirectly. When ''ftsZ'' forms a 30 subunit ring at the midpoint of the cell, it will contract and cause cell division. By expressing ''yneA'' and inhibiting ''ftsZ'' ring formation, the cells will grow filamentous. By inhibiting cell division, yneA allows the DNA damage genes to repair the DNA damage before continuing with the cell division cycle. It is hypothesized that yneA acts through an unknown transmembrane protein to inhibit ftsZ ring formation; we call this/these unknown components ???Blackbox proteins???. As the evidence shows expression of yneA leads to filamentation.
Left end of a linear transpososome sequence containing the 19 bp mosaic end in the forward orientation. The sequence also contains a PvuII cut site which releases the transpososome when cut with PvuII. Constructed compound parts intended for transposition must not contain other PvuII end, or be PCR amplified with blunt end producing enzymes (Pfu, Phusion) at the PvuII cut site between CAG and CTG. This part, and the companion part M1001 provide Biobrick tools to construct Tn5 transpososomes: linear DNA sequences which can be cut in vitro from a plasmid backbone, or created by PCR, combined in vitro with purified Tn5 trasposase protein, electroportated into cells, and finally randomly inserting into the bacterial chromosome. The parts assembled between the two mosaic ends can be delivered, including antibiotic resistance markers or other functional chromosomal insertions.
;biobrick left end, TA cloning overhang GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ;biobrick right end, TA cloning overhang GTT TCT TCC TGC AGC GGC CGC TAC TAG TA ;mosaic end with cag upstream to insert PvuII cut site cagCTGTCTCTTATACACATCT ;mosaic end reverse complement with ctg upstream to insert PvuII cut site AGATGTGTATAAGAGACAGctg ;mosaic left end in biobrick form construction primer F GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG cagCTGTCTCTTATACACATCT, MEL-F ;mosaic left end in biobrick form construction primer R GTT TCT TCC TGC AGC GGC CGC TAC TAG TA AGATGTGTATAAGAGACAGctg, MEL-R ;mosaic right end in biobrick form construction primer F GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG AGATGTGTATAAGAGACAGctg, MER-F ;mosaic right end in biobrick form construction primer R GTT TCT TCC TGC AGC GGC CGC TAC TAG TA cagCTGTCTCTTATACACATCT, MER-R
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.
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
The Estrogen Responsive Element was taken from the Human Estrogen Responsive Element
This composite part contains LacIQ (BBa_I14032) and Estrogen Receptor Element (ERE) and TetR (BBa_C0040). The Estrogen Responsive element is to be used in conjunction with ER (Estrogen Receptor). This design involves ER binding to the ERE and disrupting the constituative promoter LacIQ from producing TetR via physically getting in the way of the polymerase.
There is some data that suggests the ER is toxic to the cell. However in our experiments while we did see a decrease in growth rate we did not find ER to be fatal to the cell.
This part when used will allow the production of TetR to increase the production of BisdA and BisdB. When produced BisdA and BisdB will cause the degredation of Bisphenol A.
When used in conjunction with BisdB this enzyme can be used to degrade Bisphenol A. Activity has previously been recorded in E.coli by Miho Sasaki1 et al. 2005. Miho Sasaki1, Jun-ichi Maki, Ko-ichi Oshiman, Yoshinobu Matsumura1, and Tetsuaki Tsuchido.2005, Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp. strain AO1. Biodegredation 16: 449-459
These parts where designed to include a his tag to make it easier to view enzyme presence
When used in conjunction with BisdA this enzyme can be used to degrade Bisphenol A. Activity has previously been recorded in E.coli by Miho Sasaki1 et al. 2005. Miho Sasaki1, Jun-ichi Maki, Ko-ichi Oshiman, Yoshinobu Matsumura1, and Tetsuaki Tsuchido.2005, Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp. strain AO1. Biodegredation 16: 449-459
When used with BisdA the degredation of Bisphenol A sharply increases
Conversion of the iGEM parts registry to SBOL2.1