Difference between revisions of "Part:BBa K3431000"
(→Characterization of the activity) |
(→Characterization of the activity) |
||
| (11 intermediate revisions by 3 users not shown) | |||
| Line 5: | Line 5: | ||
===Introduction=== | ===Introduction=== | ||
Invertase, also called β-D-fructofuranosidas, is an enzyme for the hydrolysis of sucrose into glucose and fructose. It is commonly used as reporter proteins since its product, | Invertase, also called β-D-fructofuranosidas, is an enzyme for the hydrolysis of sucrose into glucose and fructose. It is commonly used as reporter proteins since its product, | ||
| − | glucose can be easily detected with a personal glucosemeter(PGM) 1 , which has a high usage rate in the public. According to the previous research, Thermotoga Maritima Invertase (invertase from Thermotoga maritima) (TmINV) has been proven to have high activity and thermo-stability compared to the commonly used commercial yeast invertase. | + | glucose can be easily detected with a personal glucosemeter(PGM) <sup>[1]</sup>, which has a high usage rate in the public. According to the previous research, <i>Thermotoga Maritima</i> Invertase (invertase from <i>Thermotoga maritima</i>) (TmINV) has been proven to have high activity and thermo-stability compared to the commonly used commercial yeast invertase<sup>[2]</sup>. |
| − | + | ||
===Characterization of the activity=== | ===Characterization of the activity=== | ||
| − | The 2020 iGEM CSMU_Taiwan has used both models and experiments to measure the activity of invertase. For the modelling, we predicted the kinetics of the invertase with MATLAB. To see the detailed modelling result, please check our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model As for the wet lab experiments, we produced the invertase with the PURExpress protein synthesis kit. Then we measured its reaction velocity under different sucrose concentrations. The invertase enzymatic reaction was executed at 55℃, which is the best activity temperature for commercial yeast invertase commonly used in PGM-based reaction 1 2 . The result of the model and the experiment is shown below. | + | The 2020 iGEM CSMU_Taiwan has used both models and experiments to measure the activity of invertase. For the modelling, we predicted the kinetics of the invertase with MATLAB. To see the detailed modelling result, please check our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model As for the wet lab experiments, we produced the invertase with the PURExpress protein synthesis kit. Then we measured its reaction velocity under different sucrose concentrations. The invertase enzymatic reaction was executed at 55℃, which is the best activity temperature for commercial yeast invertase commonly used in PGM-based reaction <sup>1, 2</sup> . The result of the model and the experiment is shown below. |
<html> | <html> | ||
<br> | <br> | ||
<div style="width=100%; display:flex; align-items: center; justify-content: center"> | <div style="width=100%; display:flex; align-items: center; justify-content: center"> | ||
| − | <img src="https://static.igem.org/mediawiki/parts/ | + | <img src="https://static.igem.org/mediawiki/parts/e/ef/T--CSMU_Taiwan--ES-concentration.jpeg" style="width:70%"> |
</div> | </div> | ||
| − | + | Figure 1. The initial velocity of the invertase enzymatic reaction under different sucrose | |
concentrations. The green line refers to the regression curve of experimental data, and the | concentrations. The green line refers to the regression curve of experimental data, and the | ||
blue line refers to the invertase activity model. | blue line refers to the invertase activity model. | ||
<br> | <br> | ||
</html> | </html> | ||
| − | + | <br> | |
<b>Results</b> The initial velocity of the reaction increases as the concentration of the substrate (sucrose) rises. The trend of experimental data fitted our model. | <b>Results</b> The initial velocity of the reaction increases as the concentration of the substrate (sucrose) rises. The trend of experimental data fitted our model. | ||
===References=== | ===References=== | ||
1. Xiang, Y., & Lu, Y. (2011). Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nature chemistry, 3(9),697–703. | 1. Xiang, Y., & Lu, Y. (2011). Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nature chemistry, 3(9),697–703. | ||
| − | https://doi.org/10.1038/nchem.1092 | + | https://doi.org/10.1038/nchem.1092<br> |
2. Du, Y., Hughes, R. A., Bhadra, S., Jiang, Y. S., Ellington, A. D., & Li, B.(2015). A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers. Scientific reports, 5, 11039. https://doi.org/10.1038/srep11039 | 2. Du, Y., Hughes, R. A., Bhadra, S., Jiang, Y. S., Ellington, A. D., & Li, B.(2015). A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers. Scientific reports, 5, 11039. https://doi.org/10.1038/srep11039 | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 15:59, 27 October 2020
Thermotoga maritima Invertase
Introduction
Invertase, also called β-D-fructofuranosidas, is an enzyme for the hydrolysis of sucrose into glucose and fructose. It is commonly used as reporter proteins since its product, glucose can be easily detected with a personal glucosemeter(PGM) [1], which has a high usage rate in the public. According to the previous research, Thermotoga Maritima Invertase (invertase from Thermotoga maritima) (TmINV) has been proven to have high activity and thermo-stability compared to the commonly used commercial yeast invertase[2].
Characterization of the activity
The 2020 iGEM CSMU_Taiwan has used both models and experiments to measure the activity of invertase. For the modelling, we predicted the kinetics of the invertase with MATLAB. To see the detailed modelling result, please check our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model As for the wet lab experiments, we produced the invertase with the PURExpress protein synthesis kit. Then we measured its reaction velocity under different sucrose concentrations. The invertase enzymatic reaction was executed at 55℃, which is the best activity temperature for commercial yeast invertase commonly used in PGM-based reaction 1, 2 . The result of the model and the experiment is shown below.
Results The initial velocity of the reaction increases as the concentration of the substrate (sucrose) rises. The trend of experimental data fitted our model.
References
1. Xiang, Y., & Lu, Y. (2011). Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nature chemistry, 3(9),697–703.
https://doi.org/10.1038/nchem.1092
2. Du, Y., Hughes, R. A., Bhadra, S., Jiang, Y. S., Ellington, A. D., & Li, B.(2015). A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers. Scientific reports, 5, 11039. https://doi.org/10.1038/srep11039
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1078
Illegal BamHI site found at 1208
Illegal XhoI site found at 1279 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 879
- 1000COMPATIBLE WITH RFC[1000]