Difference between revisions of "Part:BBa K2213003"

The part was over expressed in a BL21 (DE3) strain of E. coli and successfully purified to near homogeneity using Ni-NTA affinity media (Figure 2).

Figure 2: SDS-PAGE analysis of the different stages of purification for the cgPPK construct

Assessing the thermal stability of cgPPK2 and PduD(1-20)-cgPPK2-mCherry
The heat stability of these constructs was determined through the use of a Thermal Shift Assay of the constructs cgPPK2_His6 (https://parts.igem.org/Part:BBa_K2213003) and PduD(1-20)_mCherry_cgPPK2_His6 (https://parts.igem.org/Part:BBa_K2213005).

Figure 3: A thermal shift assay of the constructs BBa_K2213003 and BBa_K2213005 using the dye sypro orange. Readings were taken at 0.4ºC intervals, 30 seconds after the solution had maintained that temperature. The samples were tested in duplicate before being normalised, so that the highest reading of each run was equal to 1; the mean was then plotted. Error bars showing the standard deviation are also shown.

From figure 3, cgPPK2His6 showed maximum change in denatured protein between 29.8ºC - 30.6ºC; whereas the tag-mcherry-ppk construct showed peaks at 32.2ºC - 32.4ºC. This suggests that the addition of a PduD tag and mCherry protein slightly increases the heat stability of the cgPPK2 protein. The tag-mCherry-PPK construct also consistently showed a second peak at 95.2ºC. Using Imperial2011's experience with BBa_I13521 (https://parts.igem.org/Part:BBa_I13521:Experience) we believe this peak is likely to be caused by the unfolding of the mCherry domain. Further research also seems to support this hypothesis. (Probing the StaBIlity of Fluorescent Proteins by Terahertz Spectroscopy, 2014)

References

Lindner, S., Vidaurre, D., Willbold, S., Schoberth, S. and Wendisch, V. (2007). NCgl2620 Encodes a Class II Polyphosphate Kinase in Corynebacterium glutamicum. Applied and Environmental Microbiology, 73(15), pp.5026-5033.

Probing the StaBIlity of Fluorescent Proteins by Terahertz Spectroscopy. (2014). [ebook] NY: University of Buffalo, University of Colorado, pp.1-2. Available at: http://www.irmmw-thz2014.org/sites/default/files/W3_C-14.11_Xu.pdf

Contributions

Designed by: Yue Qin Group: iGEM18_WHU-China
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 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. We performed colony PCR to proof the exist of PPK in our transformed bacteria and by chance,found that WT strain may contain PPK gene as well but with a lower copy number. The blank control group indicated that there is no pollution in the PCR system. This may explain why the function of ppk is not obvious.(the red arrow shows the ppk may be in the WT strain)