Coding

Part:BBa_K3941000

Designed by: Begüm Esra Aytan   Group: iGEM21_Saint_Joseph   (2021-07-26)
Revision as of 19:29, 13 October 2021 by Boraciner (Talk | contribs)


CelAB


Summary

BBa_K3941000 is a codon-optimized version of the CelAB's catalytic region's gene. We optimized the sequence for expression and added a 6XHis at the end. It is created to produce an endo-1, 4-β-D glucanase protein to degrade cellulose and its derivatives. ­


800px-T--Saint_Joseph--Diagram-CelAB.png

Figure 1: Codon optimized CelAB and a His-Tag


Introduction

CelAB, a homolog of CelA, produced by T. turnerae T7902, an endosymbiont of the shipworm Lyrodus pedicellatus. Substrate specificity, binding properties, and cleavage products of purified CelAB were examined to evaluate its potential multiple enzymatic activities and carbohydrate binding affinities.[1]


We took the catalytic region of the original CelAB gene to use in our experiments later on optimized it to use in E. coli DH5⺠bacteria. We also used E. coli BL21 to express our plasmids.

To harvest our proteins, we added a His-tag (6XHis) at the end of our AA sequence.


Results

At first we have done a nano-drop analysis. After seeing that the numbers were too high.


T--Saint_Joseph--Nanodrop-CelAB.png

Figure 2: Nano-drop results of 3 CelABs


We wanted to be sure that there were no confounding variables, so we have done an agarose gel electrophoresis.


T--Saint_Joseph--Part-Agarose.png

Figure 3: Results of gel electrophoresis under UV. A comparison of backbone and CelAB is visible.


References

[1] Ekborg, Nathan & Morrill, Wendy & Burgoyne, Adam & Li, Li & Distel, Daniel. (2008). CelAB, a Multifunctional Cellulase Encoded by Teredinibacter turnerae T7902T, a Culturable Symbiont Isolated from the Wood-Boring Marine Bivalve Lyrodus pedicellatus. Applied and environmental microbiology. 73. 7785-8. 10.1128/AEM.00876-07. 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
    COMPATIBLE WITH RFC[1000]


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