Difference between revisions of "Part:BBa K2711000:Design"
 
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===Design Notes===
 
===Design Notes===
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Sequence had to be codon-optimized due to difficulty of synthesizing.
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In order for the dCas9/ split β-lactamase to detect a specific DNA sequence, the C. albicans DNA needs to be free in solution. During our literature search we found that the cell wall of C. albicans consists approximately of 80-90 % carbohydrate, with the three most prominent groups being β-glucan, chitin, and mannan. β-glucan and chitin are the components that form the rigidly and strength to the cell wall. β-glucans make up 47-60 % of the weight of the cell wall, and thus form the main structural component. In C. albicans the β-glucans are linked by either β-1,3 or β-1,6 bonds [2]. In addition, some bacteria may also produce beta-lactamase, making a selective lysis important. We therefore decided to use a β-1,3-glucanase and a mannanase to selectively lyse the yeast cell wall, and not that of bacteria.. Neither the glucanase nor the mannanase we needed were in the iGEM Parts Registry, so we decided to make these as new BioBricks.
  
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The glucanase was synthesized by Twist Bioscience, and the sequence was codon optimized for Escherichia coli using their suggested optimization tool.
  
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Twist graciously offered to sponsor the synthesis of the glucanase. Unfortunately, the mannanase could not be synthesized, so we continued using only the glucanase.
  
 
===Source===
 
===Source===

Latest revision as of 14:00, 14 October 2018


Glucanase


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 958
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In order for the dCas9/ split β-lactamase to detect a specific DNA sequence, the C. albicans DNA needs to be free in solution. During our literature search we found that the cell wall of C. albicans consists approximately of 80-90 % carbohydrate, with the three most prominent groups being β-glucan, chitin, and mannan. β-glucan and chitin are the components that form the rigidly and strength to the cell wall. β-glucans make up 47-60 % of the weight of the cell wall, and thus form the main structural component. In C. albicans the β-glucans are linked by either β-1,3 or β-1,6 bonds [2]. In addition, some bacteria may also produce beta-lactamase, making a selective lysis important. We therefore decided to use a β-1,3-glucanase and a mannanase to selectively lyse the yeast cell wall, and not that of bacteria.. Neither the glucanase nor the mannanase we needed were in the iGEM Parts Registry, so we decided to make these as new BioBricks.

The glucanase was synthesized by Twist Bioscience, and the sequence was codon optimized for Escherichia coli using their suggested optimization tool.

Twist graciously offered to sponsor the synthesis of the glucanase. Unfortunately, the mannanase could not be synthesized, so we continued using only the glucanase.

Source

Cellulosimicrobium cellulans

References

1. Deepak Mudgil, in Dietary Fiber for the Prevention of Cardiovascular Disease, 2017

2. Salazar et al., 2001. Overproduction, Purification, and Characterization of β-1,3- Glucanase Type II in Escherichia coli. Protein Expression and Purification 23, 219–225.

3. Oda et al., 2017. Structural and thermodynamic characterization of endo-1,3-β-glucanase: Insights into the substrate recognition mechanism. BBA - Proteins and Proteomics 1866 (2018) 415–425.