Difference between revisions of "Part:BBa K2066002"

Revision as of 02:16, 29 October 2016

Tet Monomer A w/ 8 bp spacer for ICA

Part of a suite of Iterative Capped Assembly (ICA) parts that can be used to assemble a TetO or LacO binding array of any size. These parts were designed based upon the ICA method of assembling repeat sequences from Briggs, et al. 2012 (“Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers”). These assembled binding arrays can be used to horizontally shift the slope of a transfer function through molecular titration of transcription factors (LacI or TetR).

Additionally, there are 3 different verions of tetO ICA parts in this library, with either 8,16 or 64 base pair spacers between the tetO monomers, this is because it has been suggested by Amit et al. 2012 (“Building Enhancers from the Ground Up: A Synthetic Biology Approach” that anticooperativity plays a role in the effectivity of DNA protein binding. Anticoopritivity means that if two repressor binding sites are very close to each other, a repressor binding to one can spatially hinder, or even completely prevent a repressor from binding to the other. We thought that enabling another level of tuning to allow for even finer levels of shift magnitude by altering the level of anticooperativity would be a useful tool to include within the binding array section of the toolbox. ICA method and sequence design based on Briggs et al., 2012 and its supplement.

Protocol for Iterative Capped Assembly available at: https://static.igem.org/mediawiki/parts/d/df/T--William_and_Mary--ICA.pdf

Briggs, A. W., Rios, X., Chari, R., Yang, L., Zhang, F., Mali, P., & Church, G. M. (2012). Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic acids research, gks624.

The UNS2, UNS3, UNS 4, and UNS 5 sequences are taken from Torella et al. 2013 and are ideal for cloning long sequences of monomers.

Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, gkt860.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 107

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2066002 short</partinfo>
-The part is flanked by UNS 2 and UNS 3 as per the WM UNS gibson cloning standard. This part is the TetO, 8BP spacer 'A' monomer for use in ICA to create repeated TetO Arrays of variable length. The monomer can be PCR amplified using UNS 4 and UNS 5 PCR landing pads. Next, the part should cut with BsmBI restriction enzyme to expose sticky ends and used to extend monomer in ICA. Monomer A (this part) can bind to the initiator sequence (available on our wiki) or Monomer C.  This part contains the following: UNS 2 (WM standard gibson/amplification primer site) – UNS 4 (used to provide orthogonal amplification of monomers alone) – BsmBI site – sticky end 1 - Tet Repeat –8 bp spacer (taken as first 8 bp from UNS X) – BsmBI site – Sticky end 2 - UNS 5 (orthogonal amplification in conjunction with UNS 4) – UNS 3 (WM standard gibson/amplification primer site).
+Part of a suite of Iterative Capped Assembly (ICA) parts that can be used to assemble a TetO or LacO binding array of any size. These parts were designed based upon the ICA method of assembling repeat sequences from Briggs, et al. 2012 (“Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers”). These assembled binding arrays can be used to horizontally shift the slope of a transfer function through molecular titration of transcription factors (LacI or TetR).
+Additionally, there are 3 different verions of tetO ICA parts in this library, with either 8,16 or 64 base pair spacers between the tetO monomers, this is because it has been suggested by Amit et al. 2012 (“Building Enhancers from the Ground Up: A Synthetic Biology Approach” that anticooperativity plays a role in the effectivity of DNA protein binding. Anticoopritivity means that if two repressor binding sites are very close to each other, a repressor binding to one can spatially hinder, or even completely prevent a repressor from binding to the other. We thought that enabling another level of tuning to allow for even finer levels of shift magnitude by altering the level of anticooperativity would be a useful tool to include within the binding array section of the toolbox.
 ICA method and sequence design based on Briggs et al., 2012 and its supplement.
+Protocol for Iterative Capped Assembly available at: https://static.igem.org/mediawiki/parts/d/df/T--William_and_Mary--ICA.pdf
 Briggs, A. W., Rios, X., Chari, R., Yang, L., Zhang, F., Mali, P., & Church, G. M. (2012). Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic acids research, gks624.