Difference between revisions of "Part:BBa K1763012:Design"

 
 
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<partinfo>BBa_K1763012 short</partinfo>
 
<partinfo>BBa_K1763012 short</partinfo>
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===Design Notes===
 
===Design Notes===
−
The gene sequence for MaSp2 is particularly GC-rich, which may complicate PCR.
+
This gene sequence is NOT codon optimized for E. coli. Codon optimization would have increased the GC content to very high levels (~75% GC) that would impair the use of PCR to amplify sequences. Instead, this sequence was optimized to have a lower GC content (~59% GC). The core of the protein was assembled through the use of polymerase chain assembly (PCA) where semi-complementary oligos are extended for a few cycles. The Biobrick prefix, suffix, the BsaI recognition sites, and sticky ends were added by amplifying the core with end extension primers. These constructs were cloned into pSB1C3.
  
 +
Constructs were verified through PCR and through commercial sequencing.
  
 +
The sticky ends in the AB, BC, and CA constructs code for a valine residue (GTN). A previous biobrick that was submitted, [https://parts.igem.org/Part:BBa_K1384000 BBa_K1384000] was also designed for use with ICA but its sticky ends encoded Alanine (GCN). Characterization of the biobrick revealed non-specific annealing of the sticky ends, which prevents their effective use in ICA. In order to circumvent this problem, we re-designed the sticky ends. We chose to use valine instead due to its structural and chemical similarity to alanine, and because its codons have a lower GC content. In addition, the sticky ends for these newer constructs were carefully designed such that each version of the sticky ends had at most 2 identical bases. This allowed for increased specificity. However, please note that the C version sticky end 5'-CGTG-3' has a greatly reduced, but still present ability to bind to itself due to the GC complementarity on the ends of the overhang. This effect is not observed for sticky end A, 5'-AGTT-3' despite a similar symmetry. For more information regarding these effects, please visit the 2015 UCLA iGEM notebook entries [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/3_June_2015 here] and [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/5_June_2015 here].
  
 
===Source===
 
===Source===
  
−
This part is derived from the MaSp2 gene found in the spider N. Clavipes.
+
This core sequence of this part is derived from the Major Ampullate Spidroin (MaSp) protein consensus sequences found in the spider N. Clavipes. (Hinman and Lewis, 1992 and Xu and Lewis, 1990) The MaSp comes in two forms, MaSp1 and 2, both of which confer different properties on the final fiber. MaSp1 confers strength, and MaSp2 confers elasticity. We back-translated the protein to derive a suitable coding sequence. We optimized the coding sequence for a reduced GC content (~59% GC), rather than for the organism (~75% GC). This was done to allow for normal PCR without having excessive GC pairs.
  
 
===References===
 
===References===
 +
Briggs A., Rios X., Chari R., Luhan Y., Zhang F., Mali P., and Church G. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. ''Nucleic Acids Research''. 2012;'''40'''(15): e117
 +
 +
Hinman, M.B., Lewis, R. Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. ''J. Biol. Chem''. 1992;'''267''': 19320–19324.
 +
 +
Tokareva O., Michalczechen-Lacerda V., Rech E., and Kaplan D. Recombinant DNA production of spider silk proteins. ''Microbial Biotechnology''. 2013;'''6'''(6): 651-663
 +
 +
Xu, M., Lewis, R.V. Structure of a protein superfiber: spider dragline silk. ''PNAS'';1990:'''87''': 7120.

Latest revision as of 17:59, 18 September 2015

Major Spidroin Protein 1 (MaSp1) with Sticky Ends CA


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 114


Design Notes

This gene sequence is NOT codon optimized for E. coli. Codon optimization would have increased the GC content to very high levels (~75% GC) that would impair the use of PCR to amplify sequences. Instead, this sequence was optimized to have a lower GC content (~59% GC). The core of the protein was assembled through the use of polymerase chain assembly (PCA) where semi-complementary oligos are extended for a few cycles. The Biobrick prefix, suffix, the BsaI recognition sites, and sticky ends were added by amplifying the core with end extension primers. These constructs were cloned into pSB1C3.

Constructs were verified through PCR and through commercial sequencing.

The sticky ends in the AB, BC, and CA constructs code for a valine residue (GTN). A previous biobrick that was submitted, BBa_K1384000 was also designed for use with ICA but its sticky ends encoded Alanine (GCN). Characterization of the biobrick revealed non-specific annealing of the sticky ends, which prevents their effective use in ICA. In order to circumvent this problem, we re-designed the sticky ends. We chose to use valine instead due to its structural and chemical similarity to alanine, and because its codons have a lower GC content. In addition, the sticky ends for these newer constructs were carefully designed such that each version of the sticky ends had at most 2 identical bases. This allowed for increased specificity. However, please note that the C version sticky end 5'-CGTG-3' has a greatly reduced, but still present ability to bind to itself due to the GC complementarity on the ends of the overhang. This effect is not observed for sticky end A, 5'-AGTT-3' despite a similar symmetry. For more information regarding these effects, please visit the 2015 UCLA iGEM notebook entries [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/3_June_2015 here] and [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/5_June_2015 here].

Source

This core sequence of this part is derived from the Major Ampullate Spidroin (MaSp) protein consensus sequences found in the spider N. Clavipes. (Hinman and Lewis, 1992 and Xu and Lewis, 1990) The MaSp comes in two forms, MaSp1 and 2, both of which confer different properties on the final fiber. MaSp1 confers strength, and MaSp2 confers elasticity. We back-translated the protein to derive a suitable coding sequence. We optimized the coding sequence for a reduced GC content (~59% GC), rather than for the organism (~75% GC). This was done to allow for normal PCR without having excessive GC pairs.

References

Briggs A., Rios X., Chari R., Luhan Y., Zhang F., Mali P., and Church G. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Research. 2012;40(15): e117

Hinman, M.B., Lewis, R. Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. J. Biol. Chem. 1992;267: 19320–19324.

Tokareva O., Michalczechen-Lacerda V., Rech E., and Kaplan D. Recombinant DNA production of spider silk proteins. Microbial Biotechnology. 2013;6(6): 651-663

Xu, M., Lewis, R.V. Structure of a protein superfiber: spider dragline silk. PNAS;1990:87: 7120.