Another variant of biobricked version of Isaacs' riboregulator trans activating key, taR12, only change is in the address region.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1010</bb_part>'s mRNA transcript. Part of the XOR gate comprised of <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_I1020</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1033</bb_part> and <bb_part>BBa_I1023</bb_part>).
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Case, C., Roels, S., Jense, P., Lee, J., Kleckner, N. and Simons, R. (1989). The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO 8(13): 4297-4305. 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. Lutz, R., and Bujard, H. (1997). Independent and tight regulation of transcriptional units in E. coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25(6): 1203-1210. Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO 9(4):1267-1274. E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html. References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Case, C., Roels, S., Jense, P., Lee, J., Kleckner, N. and Simons, R. (1989). The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO 8(13): 4297-4305. 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. Lutz, R., and Bujard, H. (1997). Independent and tight regulation of transcriptional units in E. coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25(6): 1203-1210. Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO 9(4):1267-1274. E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html. Complementary to beginning of <bb_part>BBa_I1010</bb_part> transcript covering 5' region, RBS, start codon, and 5 bp into coding sequence. Secondary structure designed for the IS10 anti-sense mRNA mechanism (See below). Can be used with all parts of the BBa_I system. It inhibits BBa_I1010. Incompatible with systems containing cI (wild type)
RNA Regulation Target ( Promoter driven by C0040 and cI mod #1 CDS)
Coding region for the tetR promoter (<bb_part>BBa_C0040</bb_part>) with antisense binding region for <bb_part>BBa_I1031</bb_part> and <bb_part>BBa_I1032</bb_part>, and a codon modified cI(1) protein with an LVA degradation tail.
<bb_part>BBa_I1030</bb_part> Custom cI Protein with TetR Promoter is based on BioBricks <bb_part>BBa_C0050</bb_part> and <bb_part>BBa_R0040</bb_part>. It is designed such that <bb_part>BBa_I1031</bb_part> and <bb_part>BBa_I1032</bb_part>can be used to interfere with the <bb_part>BBa_1030</bb_part> transcript by anti-sense mRNA binding, in the KISS or micRNA method shown below. <bb_part>BBa_I1030</bb_part> and <bb_part>BBa_I1031</bb_part> or <bb_part>BBa_I1032</bb_part> can act indepently of related biobrick parts <bb_part>BBa_I1041</bb_part>, <bb_part>BBa_I1042</bb_part>, <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_BBa_I1023</bb_part> and <bb_part>BBa_1013</bb_part>. [KEIL96] Incompatible with systems containing IPTG, or cI. May be some cross talk with systems containing <bb_part>BBa_I1010</bb_part>. Compatible with all other BBa_line I-line parts. Proper operation requires AMP/CAP complex, which should normally be present in the cell.
CI(1) "KISS" asRNA without promoter
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1010</bb_part>'s mRNA transcript. Has tetR promoter <bb_part>BBa_R0040</bb_part>. Part of the XOR gate comprised of <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_I1020</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1011</bb_part> and <bb_part>BBa_I1021</bb_part>), and the LacO-1 and TetR <bb_part>BBa_R0040</bb_part> promoters.
References (unparsed) here: Coleman, J., et al. Nature. (1985) 315, 601-3. Pestka, S., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 7525-28. References (unparsed) here: Coleman, J., et al. Nature. (1985) 315, 601-3. Pestka, S., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 7525-28. Complementary to beginning of <bb_part>BBa_I1010</bb_part> transcript covering RBS, start codon, and 73 bp into coding sequence. No designed secondary structure. 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. Incompatible with systems containing <bb_part>BBa_I1012</bb_part>, <bb_part>BBa_I1013</bb_part>. Compatible with <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1021</bb_part>, <bb_part>BBa_I1022</bb_part>, <bb_part>BBa_I1023</bb_part>.
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1030</bb_part>'s mRNA transcript. Has LacO-1 regulatory region with promoter. Part of the XOR gate comprised of <bb_part>BBa_I1030</bb_part> and <bb_part>BBa_I1040</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1011</bb_part> and <bb_part>BBa_I1031</bb_part>).
Complementary to beginning of <bb_part>BBa_I1030</bb_part> transcript covering junk region, RBS, start codon, and 73 bp into coding sequence. No designed secondary structure. [KEIL96] Incompatible with systems containing <bb_part>BBa_I1032</bb_part> Compatible with <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1021</bb_part>, <bb_part>BBa_I1022</bb_part>, <bb_part>BBa_I1023</bb_part>.
Grace Kenney, Neelaksh Varshney, Samantha Sutton , Daniel Shen
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1020</bb_part>'s mRNA transcript. Part of the XOR gate comprised of <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_I1020</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1013</bb_part> and <bb_part>BBa_I1023</bb_part>).
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Case, C., Roels, S., Jense, P., Lee, J., Kleckner, N. and Simons, R. (1989). The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO 8(13): 4297-4305. 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. Lutz, R., and Bujard, H. (1997). Independent and tight regulation of transcriptional units in E. coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25(6): 1203-1210. Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO 9(4):1267-1274. E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html. References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Case, C., Roels, S., Jense, P., Lee, J., Kleckner, N. and Simons, R. (1989). The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO 8(13): 4297-4305. 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. Lutz, R., and Bujard, H. (1997). Independent and tight regulation of transcriptional units in E. coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25(6): 1203-1210. Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO 9(4):1267-1274. E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html. Complementary to beginning of <bb_part>BBa_I1020</bb_part> transcript covering 5' region, RBS, start codon, and 5 bp into coding sequence. Secondary structure designed for the IS10 anti-sense mRNA mechanism (See below).
HH117a mutation specific to I1020 and I1023
Antiswitch repressing production of EYFP ("Off" switch)
The presence of theophylline prevents translation of mRNA transcribed from Biobrick BBa_E0032. The absence of theophylline allows expression of the coding region of BBa_E0032.
Transcription of the antiswitch is repressed in the presence of tetR
Adaptation of antiswitch design "S1" by Bayer and Smolke; article available at http://www.nature.com/nbt/journal/v23/n3/full/nbt1069.html
R0010 with Anti-Sense Theophillline Induced Antiswitch ("On Switch")
This part contains the R0010 Promoter region attached to an anti-sense antiswitch sequence for the regulation of EYFP This Promoter and Antiswitch construct is designed so that it can be attached to any form of the EYFP coding region. The purpose of this piece is to regulate the expression of YFP in the presence of theophylline. In this construct, the presence of theophylline causes the antiswitch, which is initially bound to the YFP coding region (preventing expression), to release and fold up on itself allowing for EYFP expression.
Adaptation of antiswitch design "S8" by Bayer and Smolke; article available at http://www.nature.com/nbt/journal/v23/n3/full/nbt1069.html
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1040</bb_part> mRNA transcript. Has tetR regulatory region and promoter. Part of the XOR gate comprised of <bb_part>BBa_I1030</bb_part> and <bb_part>BBa_I1040</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1031</bb_part> and <bb_part>BBa_I1041</bb_part>).
Complementary to beginning of <bb_part>BBa_I1040</bb_part> transcript covering junk region, RBS, start codon, and 73 bp into coding sequence. No designed secondary structure. [KEIL96] Incompatible with systems containing <bb_part>BBa_I1042</bb_part> Compatible with <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1021</bb_part>, <bb_part>BBa_I1022</bb_part>, <bb_part>BBa_I1023</bb_part>.
Samantha Sutton, Neelaksh Varshney, Daniel Shen, Grace Kenney
Coding sequence for modified cI protein with LacO-1 promoter for antisense IS10 mRNA regulation.
<bb_part>BBa_I1020</bb_part> Custom cI Protein with LacO-1 Promoter is based on BioBricks <bb_part>BBa_C0050</bb_part> and <bb_part>BBa_R0011</bb_part>. It is designed such that <bb_part>BBa_I1023</bb_part> can be used to interfere with the <bb_part>BBa_1020</bb_part> transcript by anti-sense mRNA binding, in the IS10 method shown below. <bb_part>BBa_I1020</bb_part> and <bb_part>BBa_I1023</bb_part> can act indepently of related biobrick parts <bb_part>BBa_1010</bb_part>, <bb_part>BBa_1011</bb_part>, <bb_part>BBa_1012</bb_part>, <bb_part>BBa_I1030</bb_part>, <bb_part>BBa_I1031</bb_part>, <bb_part>BBa_I1032</bb_part>, <bb_part>BBa_I1041</bb_part>, <bb_part>BBa_I1042</bb_part>, <bb_part>BBa_I1060</bb_part>, <bb_part>BBa_I1063</bb_part> and <bb_part>BBa_1013</bb_part>. Can be used with all parts of the BBa_I system. It is inhibited by BBa_I1023. Incompatible with systems containing cI (wild type)
Modified from BioBricks <bb_part>BBa_R0011</bb_part>, and <bb_part>BBa_C0051</bb_part>.
Biobricked version of Isaacs' riboregulator trans activating key, taR12
"On" Antiswitch and target EYFP in the same plasmid
This is an "On" antiswitch placed into the same plasmid as the target EYFP. Both have the same promoter. This will help to ensure a close ratio of Antiswitch to EYFP mRNA. This part was designed based on Smolke and Bayer (2005).
R0010 with Anti-Sense Theophillline Induced Antiswitch ("Off Switch")
This Promoter and Antiswitch construct is designed so that it can be attached to any form of EYFP or YFP coding region. The purpose of this piece is to regulate the expression of YFP in the presence of theophylline. In this construct, the presence of theophylline causes the antiswitch, which is initially bound up on itself (allowing expression), to release and unfold allowing it to bind to the YFP. This, in turn, halts the expression of YFP.
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1030</bb_part>'s mRNA transcript. Has LacO-1 regulatory region and a Promoter. Part of the XOR gate comprised of <bb_part>BBa_I1030</bb_part> and <bb_part>BBa_I1040</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1012</bb_part> and <bb_part>BBa_I1032</bb_part>).
Complementary to beginning of <bb_part>BBa_I1030</bb_part> transcript covering junk region, RBS, start codon, and 73 bp into coding sequence. Two stem loops flank the antisense region. [KEIL96] Incompatible with systems containing <bb_part>BBa_I1031</bb_part> Compatible with <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1021</bb_part>, <bb_part>BBa_I1022</bb_part>, <bb_part>BBa_I1023</bb_part>.
Variant of biobricked version of Isaacs' riboregulator cis repressed lock, crR12, only change is in the address region.
Antisense RNA w/BBa_I1020 Interference (micRNA)
Sequence for antisense RNA that interferes with BBa_I1020 mRNA using the micRNA scheme.
References (unparsed) here: Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970 Coleman, J., et al. Cell (1984) 37, 429-36 References (unparsed) here: Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970 Coleman, J., et al. Cell (1984) 37, 429-36 <bb_part>BBa_I1022</bb_part> antisense RNA is designed to interfere with <bb_part>BBa_I1020</bb_part> mRNA via the micRNA scheme (Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970). The sequence is designed to interfere exclusively with <bb_part>BBa_I1020</bb_part>, and act independently of <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_I1012</bb_part>. These parts are designed to use codon space to code for the same protein (<bb_part>BBa_I1010</bb_part> and <bb_part>BBa_1020</bb_part> code for lambda cI) while allowing for independent anti-sense regulation of the <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_1020</bb_part> transcripts via <bb_part>BBa_I1012</bb_part> and <bb_part>BBa_1022</bb_part>.
Anti-sense secondary structure adapted from: Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970
Antiswitch enabling production of EYFP ("On" switch)
The presence of theophylline allows translation of mRNA transcribed from Biobrick BBa_E0032. No theophylline present causes repression of the coding region of BBa_E0032.
Transcription of the antiswitch is repressed in the presence of tetR.
"Off" Antiswitch and target EYFP in the same plasmid
This is an "Off" antiswitch placed into the same plasmid as the target EYFP. Both have the same promoter. This will help to ensure a close ratio of Antiswitch to EYFP mRNA. This part was designed based on Smolke and Bayer (2005) antiswitch S8.
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1010</bb_part>'s mRNA transcript via micRNA mechanism (See below for details). Has tetR promoter <bb_part>BBa_R0040</bb_part>. Part of the XOR gate comprised of <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_I1020</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1012</bb_part> and <bb_part>BBa_I1022</bb_part>), and the LacO-1 and TetR <bb_part>BBa_R0040</bb_part> promoters.
References (unparsed) here: Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970. Coleman, J., et al. Cell (1984) 37, 429-36. References (unparsed) here: Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970. Coleman, J., et al. Cell (1984) 37, 429-36. Complementary to beginning of <bb_part>BBa_I1010</bb_part> transcript covering RBS, start codon, and 73 bp into coding sequence. No designed secondary structure. Complementary region is flanked by two stem loop structures, as specified by the micRNA mechanism. 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. 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. Incompatible with systems containing <bb_part>BBa_I1011</bb_part>, <bb_part>BBa_I1013</bb_part>. Compatible with <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1021</bb_part>, <bb_part>BBa_I1022</bb_part>, <bb_part>BBa_I1023</bb_part>.
Custom cI(2) Protein w/LacO-1 Promoter (Used for Anti-sense inhibition)
<bb_part>BBa_I1060</bb_part> Custom cI Protein with LacO-1 Promoter is based on BioBricks <bb_part>BBa_C0050</bb_part> and <bb_part>BBa_R0011</bb_part>. It is designed such that <bb_part>BBa_I1063</bb_part> can be used to interfere with the <bb_part>BBa_1060</bb_part> transcript by anti-sense mRNA binding, in the IS10 method shown below. <bb_part>BBa_I1060</bb_part> and <bb_part>BBa_I1063</bb_part> can act indepently of related biobrick parts <bb_part>BBa_1010</bb_part>, <bb_part>BBa_1011</bb_part>, <bb_part>BBa_1012</bb_part>, <bb_part>BBa_I1030</bb_part>, <bb_part>BBa_I1031</bb_part>, <bb_part>BBa_I1032</bb_part>, <bb_part>BBa_I1041</bb_part>, <bb_part>BBa_I1042</bb_part>, <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1023</bb_part> and <bb_part>BBa_1013</bb_part>. Can be used with all parts of the BBa_I system. It is inhibited by BBa_I1063. Incompatible with systems containing cI (wild type)
RNA Regulation Target ( Promoter driven by C0010 and cI(2) CDS)
Coding region for the LacO-1 promoter with antisense binding region for <bb_part>BBa_I1041</bb_part> and <bb_part>BBa_I1042</bb_part>, and a codon modified cI(1) protein with an LVA degradation tailCoding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator (<bb_part>BBa_R0010</bb_part>) and hybrid Pl-lac01 regulator as is used in parts <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1023</bb_part>, <bb_part>BBa_I1031</bb_part>, <bb_part>BBa_I1032</bb_part>, <bb_part>BBa_I1040</bb_part>, <bb_part>BBa_I1041</bb_part>, <bb_part>BBa_I1042</bb_part>, <bb_part>BBa_I1060</bb_part>, <bb_part>BBa_I1063</bb_part>, <bb_part>BBa_I1020</bb_part>. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, thus allowing transcription. This is the basis of an "Implies" gate.
<bb_part>BBa_I1040</bb_part> Custom cI Protein with LacO-1 Promoter is based on BioBricks <bb_part>BBa_C0050</bb_part>. It is designed such that <bb_part>BBa_I1041</bb_part> and <bb_part>BBa_I1042</bb_part> can be used to interfere with the <bb_part>BBa_1040</bb_part> transcript by anti-sense mRNA binding, in the KISS or micRNA method shown below. <bb_part>BBa_I1040</bb_part> and <bb_part>BBa_I1041</bb_part> or <bb_part>BBa_I1042</bb_part> can act indepently of related biobrick parts <bb_part>BBa_I1031</bb_part>, <bb_part>BBa_I1032</bb_part>, <bb_part>BBa_I1020</bb_part>, and <bb_part>BBa_1013</bb_part>. [KEIL96] Incompatible with systems containing IPTG, lactose, lacI, or cI. May be some cross talk with systems containing <bb_part>BBa_I1020</bb_part> or <bb_part>BBa_I1060</bb_part>. Compatible with all other BBa_line I-line parts. Proper operation requires AMP/CAP complex, which should normally be present in the cell.
Antisense RNA w/BBa_I1020 Interference (KISS)
Sequence for antisense RNA that interferes with BBa_I1020 mRNA using the KISS scheme.
<bb_part>BBa_I1021</bb_part> antisense RNA is designed to interfere with <bb_part>BBa_I1020</bb_part> mRNA with no expected secondary structure interactions (dubbed the KISS scheme for Keep is Simple Samantha). The sequence is designed to interfere exclusively with <bb_part>BBa_I1020</bb_part>, and act independently of <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_I1011</bb_part>. These parts are designed to use codon space to code for the same protein (<bb_part>BBa_I1010</bb_part> and <bb_part>BBa_1020</bb_part> code for lambda cI) while allowing for independent anti-sense regulation of the <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_1020</bb_part> transcripts via <bb_part>BBa_I1011</bb_part> and <bb_part>BBa_1021</bb_part>
Anti-sense secondary structure adapted from: Mizuno, T., Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970
CI(2) "KISS" asRNA without Promoter
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1040</bb_part> mRNA transcript. Part of the XOR gate comprised of <bb_part>BBa_I1030</bb_part> and <bb_part>BBa_I1040</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1031</bb_part> and <bb_part>BBa_I1041</bb_part>).
Promoter with Riboswitch designed to turn "ON" a protein
These two parts will hopefully be made by blue heron and be able to be ligated onto RBS, Coding, and Terminator sequences. It is predicted that they will serve to turn "ON" any protein we attach to it with increasing presence of theophylline.
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1030</bb_part>'s mRNA transcript. Part of the XOR gate comprised of <bb_part>BBa_I1030</bb_part> and <bb_part>BBa_I1040</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1011</bb_part> and <bb_part>BBa_I1031</bb_part>).
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1040</bb_part> mRNA transcript. Has tetR regulatory region with promoter. Part of the XOR gate comprised of <bb_part>BBa_I1030</bb_part> and <bb_part>BBa_I1040</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1032</bb_part> and <bb_part>BBa_I1042</bb_part>).
Complementary to beginning of <bb_part>BBa_I1040</bb_part> transcript covering junk region, RBS, start codon, and 73 bp into coding sequence. Two stem loops flank the antisense region. [KEIL96] Incompatible with systems containing <bb_part>BBa_I1041</bb_part> Compatible with <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1021</bb_part>, <bb_part>BBa_I1022</bb_part>, <bb_part>BBa_I1023</bb_part>.
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1010</bb_part>'s mRNA transcript via micRNA mechanism (See below for details). Has tetR promoter <bb_part>BBa_R0040</bb_part>. Part of the XOR gate comprised of <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_I1020</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1013</bb_part> and <bb_part>BBa_I1023</bb_part>), and the LacO-1 and TetR <bb_part>BBa_R0040</bb_part> promoters.
References (unparsed) here: Case, C., Roels, S., Jense, P., Lee, J., Kleckner, N. and Simons, R. (1989). The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO 8(13): 4297-4305. 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. Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439. 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. Lutz, R., and Bujard, H. (1997). Independent and tight regulation of transcriptional units in E. coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25(6): 1203-1210. Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO 9(4):1267-1274. E. coli codon usage table (http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html). References (unparsed) here: Case, C., Roels, S., Jense, P., Lee, J., Kleckner, N. and Simons, R. (1989). The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO 8(13): 4297-4305. 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. Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439. 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. Lutz, R., and Bujard, H. (1997). Independent and tight regulation of transcriptional units in E. coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25(6): 1203-1210. Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO 9(4):1267-1274. E. coli codon usage table (http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html). Complementary to beginning of <bb_part>BBa_I1010</bb_part> transcript covering RBS, start codon, and 73 bp into coding sequence. Secondary structure designed for the IS10 anti-sense mRNA mechanism (See below). 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. 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 <bb_part>BBa_I1011</bb_part>, <bb_part>BBa_I1012</bb_part>. Compatible with <bb_part>BBa_I1020</bb_part>, <bb_part>BBa_I1021</bb_part>, <bb_part>BBa_I1022</bb_part>, <bb_part>BBa_I1023</bb_part>.
Region which serves as basis for transcription of asRNA that binds to and inhibits <bb_part>BBa_I1060</bb_part> mRNA transcript. Part of the XOR gate comprised of <bb_part>BBa_I1010</bb_part> and <bb_part>BBa_I1060</bb_part>, their corresponding asRNA coding sequences (<bb_part>BBa_I1013</bb_part> and <bb_part>BBa_I1063</bb_part>).
Complementary to beginning of <bb_part>BBa_I1060</bb_part> transcript covering 5' region, RBS, start codon, and 5 bp into coding sequence. Secondary structure designed for the IS10 anti-sense mRNA mechanism (See below). Can be used with all parts of the BBa_I system. It inhibits <bb_part>BBa_I1060</bb_part>. Incompatible with systems containing cI (wild type)