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Team:TUDelft/Future Work
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In this project we worked towards a biothermometer, existing out of an RNA thermometer and a coupled pathway to produce color molecules. To measure the functioning of an RNA thermometer in general we used luciferase assays. In the end this provided us with unexpected problems as the lysis buffer provided with the luciferase assay kit interfered with our protein content measurements. Other lysis methods destroyed luciferase activity. | In this project we worked towards a biothermometer, existing out of an RNA thermometer and a coupled pathway to produce color molecules. To measure the functioning of an RNA thermometer in general we used luciferase assays. In the end this provided us with unexpected problems as the lysis buffer provided with the luciferase assay kit interfered with our protein content measurements. Other lysis methods destroyed luciferase activity. | ||
| - | Continuing this part of the project we would suggest using a different enzyme. This would have to be a non ''Escherichia coli'' enzyme which is easily measured, preferably in whole cells. It is tempting to use the general protein expression indicator GFP for this analysis, but this is not reliably quantifiable. Quantification is very likely to be important for these thermometer RNAs, as expression as a function of temperature will likely be a sigmoid curve. These type of thermometers have been shown to function in previous research <span id="cite_ref_1">[[Team:TUDelft/Future_Work#cite_note_1|[1]]]</span><span id="cite_ref_2">[[Team:TUDelft/Future_Work#cite_note_2|[2]]]</span> and even in research published during our project | + | Continuing this part of the project we would suggest using a different enzyme. This would have to be a non ''Escherichia coli'' enzyme which is easily measured, preferably in whole cells. It is tempting to use the general protein expression indicator GFP for this analysis, but this is not reliably quantifiable. Quantification is very likely to be important for these thermometer RNAs, as expression as a function of temperature will likely be a sigmoid curve. These type of thermometers have been shown to function in previous research <span id="cite_ref_1">[[Team:TUDelft/Future_Work#cite_note_1|[1]]]</span><span id="cite_ref_2">[[Team:TUDelft/Future_Work#cite_note_2|[2]]]</span> and even in research published during our project<span id="cite_ref_3">,[[Team:TUDelft/Future_Work#cite_note_3|[3]]]</span> and we are convinced that they can work in a biobrick environment as well. If the temperature dependent expression curves are not like an on-off switch, other systems in the registry such as the Schmitt trigger might be used for making on-off behavior sharper. |
To work further on the color pathway, first of all the genes still missing must be obtained by either DNA synthesis or by PCR on ''S. cerevisiae'' or other suitable organisms. All enzymes should preferably be tested for individual activity, which has not yet been done on the ''E. coli'' genes we provided to the registry. If enzyme activity has been confirmed, this pathway can be implemented as described by Martin ''et al.''<span id="cite_ref_4">[[Team:TUDelft/Future_Work#cite_note_4|[4]]]</span>. | To work further on the color pathway, first of all the genes still missing must be obtained by either DNA synthesis or by PCR on ''S. cerevisiae'' or other suitable organisms. All enzymes should preferably be tested for individual activity, which has not yet been done on the ''E. coli'' genes we provided to the registry. If enzyme activity has been confirmed, this pathway can be implemented as described by Martin ''et al.''<span id="cite_ref_4">[[Team:TUDelft/Future_Work#cite_note_4|[4]]]</span>. | ||
Latest revision as of 22:55, 29 October 2008
After a great summer of hard work we've achieved a lot, which is summarized here, here, here and here. Unfortunately a long summer did not provide us with enough time to finish the complete project's first draft. Therefore here's a mention of possible work for future iGEM teams to be done on the biothermometer project.
Future work towards a biothermometer
In this project we worked towards a biothermometer, existing out of an RNA thermometer and a coupled pathway to produce color molecules. To measure the functioning of an RNA thermometer in general we used luciferase assays. In the end this provided us with unexpected problems as the lysis buffer provided with the luciferase assay kit interfered with our protein content measurements. Other lysis methods destroyed luciferase activity.
Continuing this part of the project we would suggest using a different enzyme. This would have to be a non Escherichia coli enzyme which is easily measured, preferably in whole cells. It is tempting to use the general protein expression indicator GFP for this analysis, but this is not reliably quantifiable. Quantification is very likely to be important for these thermometer RNAs, as expression as a function of temperature will likely be a sigmoid curve. These type of thermometers have been shown to function in previous research [1][2] and even in research published during our project,[3] and we are convinced that they can work in a biobrick environment as well. If the temperature dependent expression curves are not like an on-off switch, other systems in the registry such as the Schmitt trigger might be used for making on-off behavior sharper.
To work further on the color pathway, first of all the genes still missing must be obtained by either DNA synthesis or by PCR on S. cerevisiae or other suitable organisms. All enzymes should preferably be tested for individual activity, which has not yet been done on the E. coli genes we provided to the registry. If enzyme activity has been confirmed, this pathway can be implemented as described by Martin et al.[4].
If both systems are functional, they can be easily coupled because of the biobrick standardisation. This would deliver the biothermometer as we designed it.
References
- ^ F. Narberhaus, T. Waldminghaus & S. Chowdhury. RNA thermometers. FEMS Microbiol Rev, 30(1):3-16, 2006. [http://www.ncbi.nlm.nih.gov/pubmed/16438677 PMID:16438677]
- ^ Saheli Chowdhury, Curdin Ragaz, Emma Kreuger, and Franz Narberhaus. Temperature-controlled Structural Alterations of an RNA Thermometer. The Journal of Biological Chemistry, 278(48):47915-47921, 2003. [http://www.ncbi.nlm.nih.gov/pubmed/12963744 PMID:12963744]
- ^ Juliane Neupert and Daniel Karcher and Ralph Bock. Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli. Nucleic Acids Research, 1-9, 2008. [http://www.ncbi.nlm.nih.gov/pubmed/18753148 PMID:18753148]
- ^ V. Martin, D. Pitera, S. Withers, J. Newman and J. Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology. 21(7):796-801, 2003. [http://www.ncbi.nlm.nih.gov/pubmed/12778056 PMID:12778056]
