Lambda phage attB site, reverse complement (B'-O-B)
malE - a monomer. This monomeric protein is used as a negative control when testing for dimerization.
Single chain fragment variable produced from the genetic information of an A10B hybridoma cell line. This scFV binds to the constant heavy chain 1 domain of rabbit IgG.
Shen, et al. "Single-chain Fragment Variable Antibody Piezoimmunosensors" Analytical Chemistry, Vol. 77, No. 3, February 1, 2005.
E coli Downstream Homologous Recombination Sequence
This is the downstream sequence of the homologous recombination site for the E coli chromosome provided by Chris Farrell. The recombination site is in between the ybhH and ybhD genes on the E coli chromosome.
E coli Upstream Homologous Recombination Sequence
This is the upstream sequence of the homologous recombination site for the E coli chromosome provided by Chris Farrell. The recombination site is in between the ybhH and ybhD genes on the E coli chromosome.
Vinay S. Mahajan, Brian Chow, Peter Carr
Generic spacer for ensuring a 70 bp distance between the end of the suffix of the BioBrick part containing the double terminator and the prefix of the BioBrick part containing the promoter of the new gene. Please, use the AlignX function of Vector NT to check for homology with the components in your plasmid before using this spacer.
The size of the spacer was choosed to meet the minimum length of a sequence that can be queried using the BLAST search engine. However, subsequences of it can be used to design shorter spacers. The sequence was selected from many more sequences randomly generated using the Random DNA Generator engine; the GC% parameter used as input was 50%. The sequences were selected based on the following constraints listed in their order of importance: the absence of any putative promoter regions, a low degree of homology with the Elowitz plasmid (whose components are widely used in our designs), no homology with other E.coli sequences as shown by BLASTN search results and the presence of a number of TAA stop codons. The second constraint was the most stringent leading to the elimination of most sequences. DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA. Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur): T->A @ 85 T->A @ 42 C->T @ 79 A->T @ 64 A->T @ 31 T->A @ 34 C->A @ 37 Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences. In the process of selecting the best sequence it appeared that a good alternative sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA. The sequence contains no putative promoters and shows moderate homology with the 5' end of the Ampicillin resistance gene. However a strong promoter sequence starts 12 bp downstream of this sequence, and therefore the sequence presented above was preferred. The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.
Randomly generated and optimized for several parameters (see Design notes).
Antirabbit IgG scFV: no Pst1 or Not1 sites
Reverse complement of P21 attB attachment site
Reverse Complement of P22 attB site (B'-O-B)
Reverse complement HK022 attB attachment site
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
This part has been optimized for anti-sense inhibition. It is inhibited by anti-sense parts <bb_part>BBa_I1011</bb_part>, <bb_part>BBa_I1012</bb_part>, and <bb_part>BBa_I1013</bb_part>. It contains the following regions: tetR promoter, anti-sense binding region, RBS, and cI protein with modified initial codons.
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Coleman, J., et al. Nature. (1985) 315, 601-3. Coleman, J., et al. Cell (1984) 37, 429-36. Pestka, S., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 7525-28. Jain, C. (1995). IS10 Antisense Control in Vivo is Affected by Mutations Throughout the Region of Complementarity Between the Interacting RNAs. J. Mol. Biol. 246:585-594. Kittle, J.D., Simons, R.W., Lee, J., and Kleckner, N. (1989). Insertion Sequence IS10 Anti-sense Pairing Initiates by an Interaction Between the 5' End of the Target RNA and a Loop in the Anti-sense RNA. J. Mol. Biol. 210:561-572. Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439. E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html. Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210 Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970. References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Coleman, J., et al. Nature. (1985) 315, 601-3. Coleman, J., et al. Cell (1984) 37, 429-36. Pestka, S., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 7525-28. Jain, C. (1995). IS10 Antisense Control in Vivo is Affected by Mutations Throughout the Region of Complementarity Between the Interacting RNAs. J. Mol. Biol. 246:585-594. Kittle, J.D., Simons, R.W., Lee, J., and Kleckner, N. (1989). Insertion Sequence IS10 Anti-sense Pairing Initiates by an Interaction Between the 5' End of the Target RNA and a Loop in the Anti-sense RNA. J. Mol. Biol. 210:561-572. Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439. E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html. Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210 Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970. This protein is built from several parts: - tetR promoter <bb_part> BBa_R0040</bb_part> - anti-sense binding region, as optimized from references above. - RBS from references above. - cI <bb_part>BBa_C0051</bb_part>) with slightly altered codons in the first 73 bases of the coding region (see codon usage table in references). Anti-sense The success of this system clearly rests on the ability to effectively and specifically target mRNA transcripts for degradation using anti-sense RNA. While many papers, articles, and books have been written on the subject, there are no consensus anti-sense building strategies presented. We thus chose to implement three different types of antisense inhibition: KISS, micRNA, and IS10. In the description that follows, the following nomenclature will be used: target- the mRNA transcript that we wish to inhibit. anti-sense- the anti-sense molecule which will bind and inhibit target. KISS (Keep it SImple, Silly) The simplest of the three methods, this type relies on a single-stranded linear 103 bp anti-sense that is specific to the target of interest. In addition, the first 76 base pairs of the cI region of BBa_I1010 have been codon-modified to give a different sequence that codes for the same cI protein (See BBa_I1030 and I1040). BBa_I1011 contains the reverse complement of the RBS, start codon, and 76 bp region for BBa_I1010. Thus, if both BBa_I1010 and BBa_I1011 are transcribed, the transcripts will bind to each other and BBa_I1010 will not be translated. Note that BBa_I1010 already contains a regulatory region, RBS, and coding region (a terminator must be added), while BBa_I1011 does not - thus, when using this component, the appropriate regulatory region, RBS, and terminator must be added to this part. micRNA This anti-sense mechanism relies on two stem loops flanking an anti-sense sequence that is specific for the target. The function of the stem loops is to maintain the anti-sense region in a quasi-linear state. BBa_I1012 is built in this manner, with a linear region that will bind over the RBS, start codon, and 76 bp of BBa_I1010. IS10 This method is modeled after the mechanism by which IS10 inhibits production of IS10 transposase. The anti-sense strand is transcribed from the complementary strand of the target (see below), resulting in an anti-sense strand that is 115 bp long, of which 35 bp are complementary to the target. In the absense of a target, these 35 bp form a weak stem loop with the rest of the anti-sense molecule (see below). The key element of the system is the loop at the tip of this stem loop (C-G-G-C-U-U...), which is held in a linear state by the rest of the loop. Upon exposure to the target, the linear loop is able to bind to the 5' end of the target (G-C-C-G-T-T...), and initiate an energetically-favorable zipping/twisting-together of the target and the 5' end of the stem loop (see below). In other words, one side of the weakly stable anti-sense stem loop binds 35 bp of the target, to form a more stable duplex. I1010 and I1013 Biobricks part BBa_I1013 codes for the exact anti-sense stem loop used in IS10, with two base changes. The 5'-most residues from IS10 anti-sense transcript ( U-C), which do not form part of the stem loop, were changed to G-A. These two bases are reverse-complementary to the first two base pairs of the wildtype cI coding region of BBa_I1010, and thus can bind this region. The rest of the stem loop is wild-type. The BBa_1010 transcript is targeted by BBa_I1013. The first 35 bases at the 5' end of BBa_I1010 are identical to the first 35 bases at the 5' end of the wild type target, with two differences. Note that three bases T-G-C (which code for cysteine) have been inserted at the 5' end of the cI coding region immediately after the start codon. This allows us to use a wild-type binding pattern at the base of the stem. Since this cysteine is added to the N-terminus of cI, it is not expected to alter the repression ability of cI. Incompatible with systems containing cI (wild type). Compatible with systems containing <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1021</bb_part>, <bb_part>BBa_I1022</bb_part>, <bb_part>BBa_I1023</bb_part>.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210 A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
This message system consists of a unique designated message sequence, being inserted with specific repeat sequence for recognition site by Rci site-specific recombinase.
This DNA sequence is acting as a template, to guide the make of a message inserted with specific repeat sequence for recognition by Rci recombinase in order to accomplish random rearrangement of message in encryption process.
This artificial DNA sequence is converted from text with the use of ASCII system. The specific repeat sequence "repeat 1" and "repeat 2" are come from transfer region in R64 plasmid, with genomic assession no.:AB027308.1
trkD, a functional Kup (formerly TrkD) system took up Cs+ with a moderate rate and affinity
Escherichia coli cells which contain a functional Kup (formerly TrkD) system took up Cs' with a moderate rate and affinity. Kup is a separate K+ uptake system with relatively little discrimination in the transport of the cations K+, Rb+, and Cs'. Regardless of the presence or absence of Kup, K+-replete cells took up Cs'primarily by a very low affinity mode, proportional to the ratio of the Cs' and K+ concentrations in the medium. In our project, trkD is used to collect Cs-137 when recN is activated by radiation and start the transcription of trkD,in order to absorb this kind of radioactive source like Cs-137