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Team:Potsdam Bioware/Project/Details Microviridin
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==Microviridin== | ==Microviridin== | ||
=== Modularization of the ''mdn'' gene cluster === | === Modularization of the ''mdn'' gene cluster === | ||
| + | Using two vectors (pARW071 and pARW089, respectively) containing the mdn biosynthetic gene cluster, we designed PCR primers for the amplification of the mdn genes. Using these primers, we obtained the gene fragments of mdnA, mdnB, mdnC, mdnD, mdnE and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our biobricks comply with the BioBrick standards. | ||
| + | |||
| + | The image shows the generic construction of the mdnA-biobrick. | ||
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| + | Microviridin production in E. coli cells expressing the mdn genes was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min. (reference von Elke mit angeben!?) | ||
| + | All HPLC chromatograms of the mdn-cluster extracts showed reliable peaks with a retention time of 5 min together with a number of following minor peaks. | ||
=== Generating a ''mdnA'' gene libraries === | === Generating a ''mdnA'' gene libraries === | ||
=== Expression Backbones === | === Expression Backbones === | ||
Revision as of 20:31, 21 September 2011
Contents |
Microviridin
Modularization of the mdn gene cluster
Using two vectors (pARW071 and pARW089, respectively) containing the mdn biosynthetic gene cluster, we designed PCR primers for the amplification of the mdn genes. Using these primers, we obtained the gene fragments of mdnA, mdnB, mdnC, mdnD, mdnE and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our biobricks comply with the BioBrick standards.
The image shows the generic construction of the mdnA-biobrick.
Microviridin production in E. coli cells expressing the mdn genes was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min. (reference von Elke mit angeben!?) All HPLC chromatograms of the mdn-cluster extracts showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.
