Difference between revisions of "Part:BBa K3941000"

Revision as of 18:50, 17 October 2021

CelAB

Summary

BBa_K3941000 is a codon-optimized (for E. coli DH5⍺) version of the CelAB's catalytic region's sequence. 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. ­

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]

Design

We took the catalytic region of the original CelAB sequence to use in our experiments and later on optimized it to use in E. coli DH5⍺ 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 spectrophotometer absorbance analysis. After seeing that the numbers were too high.

Figure 2: Spectrophotometer results of 3 CelABs

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

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

Lastly we conducted a CMCase Activity Analysis. CelAB got an absorbance of 0,043 and enzyme activity of 1.4e-2 umol/L.min. We use enzyme absorbance to calculate our enzyme activity and the activity of the CelAB wasn’t the highest of three but it was pretty good for us.

Figure 4: Calibration Curve for CMCase Activity Analysis

Figure 5: Graphs of CelAB's CMCase Activity Analysis

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