Part:BBa_K2132004
HydA with SpyCatcher, Histag and TEV site
This part encodes the major enzyme of [FeFe] hydrogenase HydA is the main catalytic unit is a modified and codon-optimized protein, we added Histag and TEV site to the N-terminal site of HydA, and SpyCatcher to the C-terminal site of HydA.
Also, we optimized [FeFe] Hydrogenases (HydA) originally from the bacterium Clostridium acetobutylicum (Original coding sequence: hydA, BBa_K535002, Designed by: iGEM11_UNAM-Genomics_ Mexico.) to accept electrons and therefor enable catalytic production of hydrogen in our project. The optimized coding sequence would produce more protein, theoretically. And optimization also improved the activity of [FeFe] Hydrogenases according to the experiment that we did.
For more detail information, please visit http://2016.igem.org/Team:ShanghaitechChina/Parts and http://2016.igem.org/Team:ShanghaitechChina/Hydrogen .
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 790
Illegal AgeI site found at 1300 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2053
Illegal SapI.rc site found at 663
Illegal SapI.rc site found at 1005
Results of Cloning
We fused pACE-Histag-TEV-hydA-Spycatcher with pDK-hydF, pDC-hydE, pDS-hydG together to give the new plasmid. To test if we successfully fused the four subplasmid, we use single restricted endonuclease digestion of Nde I. The restriction gives three bands on a 1% TAE Gel, in accordance with the band predicted by SnapGene®.
Figure 1 The test of fusion of the plasmid.
Enzyme Activity Assay
Unfortunately, compared to the modified HydA protein with SpyTag, the modified HydA protein with SpyCather didn't have the activity of hydrogen production. We will improve or achieve the activity of this modified enzyme in the future work.
Figure 2 The Activity of HydA-SpyCatcher results compared with that of HydA-SpyTag.
| None |