Difference between revisions of "Part:BBa K2325103"
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| + | We digested and ligated the pGEX-2T vector and fusion protein gene and then transformed it into E.coli strain BL21(DE3). | ||
| + | We also constructed an co-expressed vector pETDuet-for-ptx where these two genes are expressed separately. | ||
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[[File:BBa_K2325103--2.png|750px]]<br /> | [[File:BBa_K2325103--2.png|750px]]<br /> | ||
Revision as of 02:42, 28 October 2017
An expression device for fusion protein of Formamidase and Phosphite dehydrogenase
The BBa_K2325001 harbors a coding sequence of and formamidase(FOR) and phosphite dehydrogenase (PTDH) with a linker between them, which can simultaneously catalyzes the conversion of formamide to ammonia and phosphite to phosphate, respectively. The device also contains GST tag for increasing the solubility of exogenous protein.
Gel analysis
M：1kb DNA Marker P;
1: plasmid from BL21(DE3) (pGEX-for-ptx);
2&3: plasmid from BL21(DE3) (pETDuet-for-ptx).
Usage and Biology
We digested and ligated the pGEX-2T vector and fusion protein gene and then transformed it into E.coli strain BL21(DE3). We also constructed an co-expressed vector pETDuet-for-ptx where these two genes are expressed separately.
Figure 1. Growth curves of E.coli (with two ways of constructed plasmids possessing the formamidase gene and phosphite dehydrogenase gene) in a basal MOPS medium (Na2HPO3•5H20 (1.32mM) , NH3CO (200mM)).
The results showed that the growth of recombinant strains of both construction ways are encouraging, indicating the recombinant strains were able to utilize formamide and phosphite at the same time, while the negative control group was unable to grow normally due to the lack of nitrogen and phosphorus sources.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 821
Illegal NgoMIV site found at 1308
Illegal NgoMIV site found at 1503
Illegal NgoMIV site found at 1650 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 159