Difference between revisions of "Part:BBa K2789020"
| Line 5: | Line 5: | ||
This part originally came from 17_Manchester team. But we wanted to get this part and build a more complicated pathway, we found that there is no coding sequence of only PPK without any other protein. Thus we made this part and did an experiment to verify the function of it.It worked well and we found that this kind of PPK can be PCR out of E.coli.BL21. This protein actually can catalyze the formation of heterochromatic granules (Poly P) thus use the natural phosphorus transport system to accumulate the phosphorus in the bacteria. | This part originally came from 17_Manchester team. But we wanted to get this part and build a more complicated pathway, we found that there is no coding sequence of only PPK without any other protein. Thus we made this part and did an experiment to verify the function of it.It worked well and we found that this kind of PPK can be PCR out of E.coli.BL21. This protein actually can catalyze the formation of heterochromatic granules (Poly P) thus use the natural phosphorus transport system to accumulate the phosphorus in the bacteria. | ||
| − | ==Experiment to test the function of PPK== | + | ===Experiment to test the function of PPK=== |
We have created a PPK-pET28a(+) plasmid and transformed the BL21 with it. We add 100μL WT and the transformed BL21 separately into 100ml LB culture and incubate the bacteria at 37°C with 200rpm in about 20 hours. After incubation, we add IPTG with a final concentration about 0.5mM and continue the incubate for 16hours. We use the kit manufactured by HANGZHOU LOHAND BIOLOGICAL Co. Ltd. to measure the phosphorus concentration in the media. Before that ,the culture is centrifuged at 10,000×g for 3 minutes to clear the most bacteria and OD600 shows two groups has similar bacteria concentration(OD600 about 1.6). The supernatant is diluted with 99 times water to make the phosphorus suitable to measure. (3 same groups are done in the test. pET28a(+) is a expression vector that can be induced by IPTG) | We have created a PPK-pET28a(+) plasmid and transformed the BL21 with it. We add 100μL WT and the transformed BL21 separately into 100ml LB culture and incubate the bacteria at 37°C with 200rpm in about 20 hours. After incubation, we add IPTG with a final concentration about 0.5mM and continue the incubate for 16hours. We use the kit manufactured by HANGZHOU LOHAND BIOLOGICAL Co. Ltd. to measure the phosphorus concentration in the media. Before that ,the culture is centrifuged at 10,000×g for 3 minutes to clear the most bacteria and OD600 shows two groups has similar bacteria concentration(OD600 about 1.6). The supernatant is diluted with 99 times water to make the phosphorus suitable to measure. (3 same groups are done in the test. pET28a(+) is a expression vector that can be induced by IPTG) | ||
<br/> | <br/> | ||
| − | ==The protocol for measuring the total phosphorus== | + | ===The protocol for measuring the total phosphorus=== |
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Revision as of 07:55, 17 October 2018
Polyphosphokinase that can catalyze the formation of PolyP
This part originally came from 17_Manchester team. But we wanted to get this part and build a more complicated pathway, we found that there is no coding sequence of only PPK without any other protein. Thus we made this part and did an experiment to verify the function of it.It worked well and we found that this kind of PPK can be PCR out of E.coli.BL21. This protein actually can catalyze the formation of heterochromatic granules (Poly P) thus use the natural phosphorus transport system to accumulate the phosphorus in the bacteria.
Experiment to test the function of PPK
We have created a PPK-pET28a(+) plasmid and transformed the BL21 with it. We add 100μL WT and the transformed BL21 separately into 100ml LB culture and incubate the bacteria at 37°C with 200rpm in about 20 hours. After incubation, we add IPTG with a final concentration about 0.5mM and continue the incubate for 16hours. We use the kit manufactured by HANGZHOU LOHAND BIOLOGICAL Co. Ltd. to measure the phosphorus concentration in the media. Before that ,the culture is centrifuged at 10,000×g for 3 minutes to clear the most bacteria and OD600 shows two groups has similar bacteria concentration(OD600 about 1.6). The supernatant is diluted with 99 times water to make the phosphorus suitable to measure. (3 same groups are done in the test. pET28a(+) is a expression vector that can be induced by IPTG)
The protocol for measuring the total phosphorus
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 53
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 732