Difference between revisions of "Part:BBa I1013:Design"

Revision as of 02:03, 5 June 2006 (view source) Registry (Talk | contribs) ← Older edit		Latest revision as of 15:11, 21 July 2006 (view source) Smelissali (Talk | contribs) (→‎Design Notes)
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−	===Design Notes===	+	===Design Notes===
−	References (unparsed) here:	+	Complementary to beginning of <bbpart>BBa_I1010</bbpart> transcript      covering RBS, start codon, and 73 bp into coding sequence. Secondary structure designed for the IS10 anti-sense mRNA mechanism (See below).  <blockquote>        <B>Anti-sense</B>
−	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.	+	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:
−	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.	+	<em>target</em>- the mRNA transcript that we wish to inhibit.
−	Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo.      J. theor. Biol. 186: 431-439.	+	<em>anti-sense</em>- the anti-sense molecule which will bind and                    inhibit <em>target</em>.           <blockquote>
−	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.	+	<strong><em>IS10</em></strong>        <img src="http://biobricks.ai.mit.edu/IAP_Projects/YoungPower/as_IS10.gif">
−	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.	+	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.
−	Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding      at the transposase translation initiation site. EMBO 9(4):1267-1274.	+	<em>I1010 and I1013</em>
−	E. coli codon usage table (<a href="http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html">http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html</a>).	+	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.    </blockquote>                </blockquote>
		+
		+	==References==
	References (unparsed) here:		References (unparsed) here:
Line 37:		Line 40:
	Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding      at the transposase translation initiation site. EMBO 9(4):1267-1274.		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 (<a href="http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html">http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html</a>).	+	E. coli codon usage table (<a href="http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html">http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html</a>).
−		+
−	Complementary to beginning of <bbpart>BBa_I1010</bbpart> transcript      covering RBS, start codon, and 73 bp into coding sequence. Secondary structure designed for the IS10 anti-sense mRNA mechanism (See below).  <blockquote>        <B>Anti-sense</B>	+
−		+
−	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:	+
−		+
−	<em>target</em>- the mRNA transcript that we wish to inhibit.	+
−		+
−	<em>anti-sense</em>- the anti-sense molecule which will bind and                    inhibit <em>target</em>.            <blockquote>	+
−		+
−	<strong><em>IS10</em></strong>        <img src="http://biobricks.ai.mit.edu/IAP_Projects/YoungPower/as_IS10.gif">	+
−		+
−	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.	+
−		+
−	<em>I1010 and I1013</em>	+
−		+
−	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.    </blockquote>                </blockquote>	+
−		+
−	Incompatible with systems containing <bbpart>BBa_I1011</bbpart>, <bbpart>BBa_I1012</bbpart>.	+
−		+
−	Compatible with <bbpart>BBa_I1020</bbpart>, <bbpart>BBa_I1021</bbpart>, <bbpart>BBa_I1022</bbpart>, <bbpart>BBa_I1023</bbpart>.	+
−		+
−		+
−		+
	===Source===		===Source===

---

Latest revision as of 15:11, 21 July 2006

CI(1) IS10 asRNA

Design Notes

Complementary to beginning of BBa_I1010 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 <img src="http://biobricks.ai.mit.edu/IAP_Projects/YoungPower/as_IS10.gif">

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.

References

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 (<a href="http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html">http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html</a>).

Source

Custom design.

References