Difference between revisions of "Part:BBa K2789020"

Latest revision as of 08:36, 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

Kit: made by HANGZHOU LOHAND BIOLOGICAL Co. Ltd. Principle: Ammonium molybdate spectrophotometric method Steps: 1. Add one pack of Agent1 to 5ml desired liquid*.
2. Shake it thoroughly to mix the solution.
3. Put it into a autoclave setting at 121°C for 20 minutes to decompose the matter in liquid.
4. When cooling down, add a pack of Agent2 and mix it thoroughly again.
5. Add 7 drops of Activing AgentP .
6. Vortex the solution for 1 minute.
7. Use a spectrophotometer to measure OD value at 700nm.

• The liquid should be dilute to make the total phosphorus at 1-20mg/L.
  

PPK may be in the WT E.coli

We can see a 10% absorption in the experiment which may indicate the PPK works well. Additional Information We performed colony PCR to proof the exist of PPK in our transformed bacteria and found that WT may contain PPK gene as well but with a lower copy number. The blank control group indicated there is no pollution in the PCR system. This may explain why the function of ppk is not so obvious.

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