Difference between revisions of "Part:BBa K2213003"

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===Contributions===
 
===Contributions===
 
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Experiment to test the function of PPK:
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Experiment to test the function of PPK:<br/>
 
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)
 
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We can see a 10% absorption in the experiment which may indicate the PPK works well.
 
We can see a 10% absorption in the experiment which may indicate the PPK works well.
 
Additional Information
 
Additional Information
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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.<br/>
+
We performed colony PCR to proof the exist of PPK in our transformed bacteria and by chance,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 obvious.<br/>
 
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Revision as of 10:49, 17 October 2018


cgPPK2_His6

Usage and Biology


Cornybacterium glutamicum class II polyphosphate kinase (cgPPK) with a fused C terminal hexa-histidine tag to allow purification using immobilised metal ion affinity chromatography. First isolated and characterised by Lindner et al. (2007). Under the experimental conditions used by the authors, cgPPK was found to have a specific activity of 30/s. Under our own experimental conditions

ADPGlo_cgppk_size_1.5.png

Figure 1. Comparison of specific activity between the E. coli class I PPK and the Corynebacterium glutamicum class II PPK (Lindner et al., 2007)


Characterisation


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).

CgPPK_size_1.png

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).

Thermalshiftassaymanchesterjess3.png


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)



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 82
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 761

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


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 by chance,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 obvious.