Difference between revisions of "Part:BBa K1914005"
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Lysozyme C from Gallus gallus codon optomised for E.coli with a OmpA signal peptide to target the periplasm as well as a flag tag. The device is under a T7 promoter iducible using IPTG. | Lysozyme C from Gallus gallus codon optomised for E.coli with a OmpA signal peptide to target the periplasm as well as a flag tag. The device is under a T7 promoter iducible using IPTG. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| + | This device is BioBrick compatible and codon optimised for E. coli strain K12. All of our parts are under the pT7 inducible promoter (BBa_I712074) with an Elowitz ribosome binding site (BBa_B0034) before the protein coding region and a double terminator (BBa_B0015) after.We chose the parts we have used as they are either some of the most popular BioBricks used by other teams throughout the history of the iGEM competition or been awarded a registry star. One of the core aims of our project was to make it relatable and useful to as many future teams as possible. We believed that using the most popular parts on the registry reflects this intention. | ||
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| + | To show the activity of lysozyme, a molecular probes EnzCheck lysozyme assay kit from Thermo fisher scientific was used. The coding sequence (CDS) contains an OmpA signal peptide targeting it to the perisplasm therfore lysozyme will only be detectable if the cells have lysed. The kit uses a substrate containing Micrococcus lysodeikticus cell walls labelled with fluorescein to such as degree that fluorescence is quenched. The presence of lysozyme causes a sharp increase in fluorescence by easing the quenching. The increase in fluorescence is proportional to lysozyme activity in the sample. The fluorescence assay was used to measure the activity of the freshly transformed kill switch and that of the cultures grown in the ministat. CFUs were also used as a measure of efficiency by comparing number of colonies to a control. 5 ml ovenights of E. coli BL21 (DE3) transformed with pSB1C3 lysozyme were used to inoculate 250 ml Erlenmeyer flasks containing 50 ml of LB laced with 35 µg/ml chloramphenicol. Once an OD of 0.23 was reached IPTG was added to a final concentration of 0.2 nM. Protein production was allowed to proceed for 2 hrs. The sample was serially diluted (10-2,10-3,10-4). 200 µl of each dilution factor was spread plated and incubated at 37 °C overnight. CFUs were then compared to a control treated in the same way. | ||
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| + | No difference in CFUs was observed between the control and the samples producing lysozyme. The results of the EnzCheck lysozyme assay were inconclusive. | ||
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Latest revision as of 12:53, 18 October 2016
pT7 Lysozyme C E.coli codon optimised, signal peptide, flag tag
Lysozyme C from Gallus gallus codon optomised for E.coli with a OmpA signal peptide to target the periplasm as well as a flag tag. The device is under a T7 promoter iducible using IPTG.
Usage and Biology
This device is BioBrick compatible and codon optimised for E. coli strain K12. All of our parts are under the pT7 inducible promoter (BBa_I712074) with an Elowitz ribosome binding site (BBa_B0034) before the protein coding region and a double terminator (BBa_B0015) after.We chose the parts we have used as they are either some of the most popular BioBricks used by other teams throughout the history of the iGEM competition or been awarded a registry star. One of the core aims of our project was to make it relatable and useful to as many future teams as possible. We believed that using the most popular parts on the registry reflects this intention.
To show the activity of lysozyme, a molecular probes EnzCheck lysozyme assay kit from Thermo fisher scientific was used. The coding sequence (CDS) contains an OmpA signal peptide targeting it to the perisplasm therfore lysozyme will only be detectable if the cells have lysed. The kit uses a substrate containing Micrococcus lysodeikticus cell walls labelled with fluorescein to such as degree that fluorescence is quenched. The presence of lysozyme causes a sharp increase in fluorescence by easing the quenching. The increase in fluorescence is proportional to lysozyme activity in the sample. The fluorescence assay was used to measure the activity of the freshly transformed kill switch and that of the cultures grown in the ministat. CFUs were also used as a measure of efficiency by comparing number of colonies to a control. 5 ml ovenights of E. coli BL21 (DE3) transformed with pSB1C3 lysozyme were used to inoculate 250 ml Erlenmeyer flasks containing 50 ml of LB laced with 35 µg/ml chloramphenicol. Once an OD of 0.23 was reached IPTG was added to a final concentration of 0.2 nM. Protein production was allowed to proceed for 2 hrs. The sample was serially diluted (10-2,10-3,10-4). 200 µl of each dilution factor was spread plated and incubated at 37 °C overnight. CFUs were then compared to a control treated in the same way.
No difference in CFUs was observed between the control and the samples producing lysozyme. The results of the EnzCheck lysozyme assay were inconclusive.
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]