Difference between revisions of "Part:BBa K3311007"
LucyShi 2018 (Talk | contribs) |
SylviaSong (Talk | contribs) |
||
| Line 15: | Line 15: | ||
We used EMSA to verify to prove the combination of HucR and pHucO. This experiment contains two items: biotin labeled pHucO and HucR-mCherry fusion protein. The fusion protein purification is a necessary phase which is playing crucial role within the whole applied design test because our next experiment. We purified HucR-mCherry by His tag (Figure 1), more detailed description of how the protein was purified was showed in protocols in our wiki. | We used EMSA to verify to prove the combination of HucR and pHucO. This experiment contains two items: biotin labeled pHucO and HucR-mCherry fusion protein. The fusion protein purification is a necessary phase which is playing crucial role within the whole applied design test because our next experiment. We purified HucR-mCherry by His tag (Figure 1), more detailed description of how the protein was purified was showed in protocols in our wiki. | ||
| − | |||
[[File:HucR-pHucO.png|900px|thumb|left|Figure 1 (A) Photos of the protein purification process.(B)The SDS-PAGE gel of purified protein.(Notes: line1, whole bacteria; line2, supernatant; line3, flow-through; line4, 1st wash; line5, 2nd wash; line6, 1st elution; line7, 2nd elution; line8, 3rd elution)]] | [[File:HucR-pHucO.png|900px|thumb|left|Figure 1 (A) Photos of the protein purification process.(B)The SDS-PAGE gel of purified protein.(Notes: line1, whole bacteria; line2, supernatant; line3, flow-through; line4, 1st wash; line5, 2nd wash; line6, 1st elution; line7, 2nd elution; line8, 3rd elution)]] | ||
Latest revision as of 08:06, 21 October 2019
HucR-mCherry-His
The function of this composite part is express HucR-mCherry fusion protein for EMSA and applied design test.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
We used EMSA to verify to prove the combination of HucR and pHucO. This experiment contains two items: biotin labeled pHucO and HucR-mCherry fusion protein. The fusion protein purification is a necessary phase which is playing crucial role within the whole applied design test because our next experiment. We purified HucR-mCherry by His tag (Figure 1), more detailed description of how the protein was purified was showed in protocols in our wiki.
EMSA Results
The HucR-mCherry and biotin labeled pHucO were incubated together and then separated by EMSA gel. The HucR-mCherry-pHucO complex moves more slowly than non-binding labeled pHucO probes. After adding uric acid, HucR-mCherry-pHucO complex will decrease and could be viewed in EMSA gel. The concentration of HucR-mCherry increases from 0 to 1 g, which means the more HucR-mCherry is, the more the combination is, proving the combination of HucR-mCherry and pHucO (Figure 2A).
When uric acid appears in the solution, HucR-mCherry loses its function and falls off from the pHucO. Consequently, the downstream gene can be translated normally. The EMSA shows that, under the same concentration of HucR-mCherry and pHucO, there is a negative relationship between the concentration of uric acid and HucR-mCherry-pHucO complex. As the concentration of uric acid increases from 0 ~ 1000 M, the color of HucR-mCherry-pHucO complex become lighter (Figure 2B ), indicating the effect of uric acid on HucR-mCherry-pHucO complex.
