Difference between revisions of "Part:BBa K5387000:Experience"
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==Size Exclusion Chromatography== | ==Size Exclusion Chromatography== | ||
There were some shorter fragments after the purification with a Ni-NTA agarose column, hence a size exclusion chromatography was conducted to seperate the fragments. The seperation was partly succesfull, due to the three largest fragments differed too little in size to seperate completely with the columns available. | There were some shorter fragments after the purification with a Ni-NTA agarose column, hence a size exclusion chromatography was conducted to seperate the fragments. The seperation was partly succesfull, due to the three largest fragments differed too little in size to seperate completely with the columns available. | ||
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| + | <left>{{#tag:html|<img src="file1" width="400px"/>}} | ||
| + | Figure P: Before sie exclusion chromatography. | ||
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| + | <left>{{#tag:html|<img src="https://static.igem.wiki/teams/5387/a12-dtt-prom-1st.png" width="400px"/>}} | ||
| + | Figure Q: After size exclusion chromatography. | ||
==Circular Dichroism== | ==Circular Dichroism== | ||
To investigate whether the enzyme was correctly folded, a circular dichroism measurement was performed. The results from "figure C", which shows alpha-helical structure with the dips at 208 nm and 220,nm, was further analyzed with the BestSel analysis program based on the studie [1] gives an estimated secondary structure of 64,7% a-helical and 35,3% beta-sheet structure of the enzyme. The estimated structure aligns with the generated AlphaFold structure [2] of 12R-LOX with an average model confidence of more than 90%. | To investigate whether the enzyme was correctly folded, a circular dichroism measurement was performed. The results from "figure C", which shows alpha-helical structure with the dips at 208 nm and 220,nm, was further analyzed with the BestSel analysis program based on the studie [1] gives an estimated secondary structure of 64,7% a-helical and 35,3% beta-sheet structure of the enzyme. The estimated structure aligns with the generated AlphaFold structure [2] of 12R-LOX with an average model confidence of more than 90%. | ||
| − | + | <left>{{#tag:html|<img src="https://static.igem.wiki/teams/5387/a12-dtt-prom-1st.png" width="400px"/>}} | |
==NanoDSF== | ==NanoDSF== | ||
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==Wavelength Scan== | ==Wavelength Scan== | ||
| + | <left>{{#tag:html|<img src="https://static.igem.wiki/teams/5387/a12-dtt-prom-1st.png" width="400px"/>}} | ||
Revision as of 12:51, 22 September 2024
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Applications of BBa_K5387000
The enzyme 12R-LOX was succesfully expressed and multiple measurements was performed.
Size Exclusion Chromatography
There were some shorter fragments after the purification with a Ni-NTA agarose column, hence a size exclusion chromatography was conducted to seperate the fragments. The seperation was partly succesfull, due to the three largest fragments differed too little in size to seperate completely with the columns available.
<left>
Figure P: Before sie exclusion chromatography.
<left>
Figure Q: After size exclusion chromatography.
Circular Dichroism
To investigate whether the enzyme was correctly folded, a circular dichroism measurement was performed. The results from "figure C", which shows alpha-helical structure with the dips at 208 nm and 220,nm, was further analyzed with the BestSel analysis program based on the studie [1] gives an estimated secondary structure of 64,7% a-helical and 35,3% beta-sheet structure of the enzyme. The estimated structure aligns with the generated AlphaFold structure [2] of 12R-LOX with an average model confidence of more than 90%.
<left>
NanoDSF
Discovered the two Tm of the enzyme at approximately 45°C and 57°C, as seen in "figure D" below.
<left>
Wavelength Scan
<left>
References
[1] https://www.pnas.org/doi/10.1073/pnas.1500851112
[2] https://www.uniprot.org/uniprotkb/O75342/entry
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