Difference between revisions of "Part:BBa K3941000"
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<b><font size="+1">Summary</font></b> | <b><font size="+1">Summary</font></b> | ||
| − | BBa_K3941000 is a codon-optimized version of the CelAB's catalytic region's | + | BBa_K3941000 is a codon-optimized (for <i>E. coli</i> DH5⍺) version of the CelAB's catalytic region's sequence. We optimized the sequence for avoiding the codon degeneration and provide the right expression. Than we added a 6XHis at the end of the sequence. This sequence engineered to produce an endo-1, 4-β-D glucanase protein to degrade cellulose and its derivatives. |
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https://static.igem.org/mediawiki/parts/thumb/3/30/T--Saint_Joseph--Diagram-CelAB.png/800px-T--Saint_Joseph--Diagram-CelAB.png | https://static.igem.org/mediawiki/parts/thumb/3/30/T--Saint_Joseph--Diagram-CelAB.png/800px-T--Saint_Joseph--Diagram-CelAB.png | ||
| − | <font size="- | + | |
| + | <font size="-2"><b>Figure 1:</b> Codon optimized CelAB and a His-Tag</font> | ||
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CelAB, a homolog of CelA, produced by <i>T. turnerae T7902</i>, an endosymbiont of the shipworm <i>Lyrodus pedicellatus</i>. Substrate specificity, binding properties, and cleavage products of purified CelAB were examined to evaluate its potential multiple enzymatic activities and carbohydrate binding affinities.[1] | CelAB, a homolog of CelA, produced by <i>T. turnerae T7902</i>, an endosymbiont of the shipworm <i>Lyrodus pedicellatus</i>. Substrate specificity, binding properties, and cleavage products of purified CelAB were examined to evaluate its potential multiple enzymatic activities and carbohydrate binding affinities.[1] | ||
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| − | To | + | <b><font size="+1">Design</font></b> |
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| + | We took the catalytic region of the original CelAB sequence to express in <i>E.coli</i> experiments and then codon optimized it to cloning in <i>E. coli</i> DH5⍺ bacteria. We also used <i>E. coli</i> BL21 to gene expression and enzyme production. | ||
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| + | To purifying correctly our recombinant enzyme, we added a 6X His-tag at the end of our AA sequence. | ||
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<b><font size="+1">Results</font></b> | <b><font size="+1">Results</font></b> | ||
| − | At first we have done a | + | At first we have done a spectrophotometer absorbance analysis. |
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https://static.igem.org/mediawiki/parts/b/bd/T--Saint_Joseph--Nanodrop-CelAB.png | https://static.igem.org/mediawiki/parts/b/bd/T--Saint_Joseph--Nanodrop-CelAB.png | ||
| − | <font size="- | + | <font size="-2"><b>Figure 2:</b> Spectrophotometer results of 3 CelABs</font> |
We wanted to be sure that there were no confounding variables, so we have done an agarose gel electrophoresis. | We wanted to be sure that there were no confounding variables, so we have done an agarose gel electrophoresis. | ||
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https://static.igem.org/mediawiki/parts/5/53/T--Saint_Joseph--Part-Agarose.png | https://static.igem.org/mediawiki/parts/5/53/T--Saint_Joseph--Part-Agarose.png | ||
| − | <font size="- | + | <font size="-2"><b>Figure 3:</b> Results of gel electrophoresis under UV. A comparison of backbone and CelAB is visible.</font> |
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| + | Lastly we conducted a CMCase Activity Analysis. CelAB got an absorbance of 0,5765 and enzyme activity of 17,5 U/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. | ||
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| + | https://2021.igem.org/wiki/images/thumb/b/b0/T--Saint_Joseph--Standard-Glucose-Calibration-Curve.png/320px-T--Saint_Joseph--Standard-Glucose-Calibration-Curve.png | ||
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| + | <font size="-2"><b>Figure 4:</b> Calibration Curve for CMCase Activity Analysis</font> | ||
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| + | https://2021.igem.org/wiki/images/thumb/8/8b/T--Saint_Joseph--CelAB-absorbance-values.png/320px-T--Saint_Joseph--CelAB-absorbance-values.png | ||
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| + | <font size="-2"><b>Figure 5:</b> Graphs of CelAB's CMCase Activity Analysis</font> | ||
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Latest revision as of 10:48, 19 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 avoiding the codon degeneration and provide the right expression. Than we added a 6XHis at the end of the sequence. This sequence engineered 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 express in E.coli experiments and then codon optimized it to cloning in E. coli DH5⍺ bacteria. We also used E. coli BL21 to gene expression and enzyme production.
To purifying correctly our recombinant enzyme, we added a 6X His-tag at the end of our AA sequence.
Results
At first we have done a spectrophotometer absorbance analysis.
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 0,5765 and enzyme activity of 17,5 U/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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]