Difference between revisions of "Part:BBa K3595001"
Liyingying (Talk | contribs) |
Liyingying (Talk | contribs) |
||
| Line 9: | Line 9: | ||
=Usage and Biology= | =Usage and Biology= | ||
This part can be used as a coding sequence after the promoter pTac and RBS B0034. The argA protein can be translated under the induction of IPTG. We constructed plasmids pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA using argA and argAfbr, respectively. The constructed plasmid was transformed into <i>E.coli DH10b-ΔargR </i> host cell to test its ammonia degradation efficiency. | This part can be used as a coding sequence after the promoter pTac and RBS B0034. The argA protein can be translated under the induction of IPTG. We constructed plasmids pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA using argA and argAfbr, respectively. The constructed plasmid was transformed into <i>E.coli DH10b-ΔargR </i> host cell to test its ammonia degradation efficiency. | ||
| − | [[File:T--GZ_HFI--argA|600px|thumb|center|The structure of the plasmid pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA ]] | + | [[File:T--GZ_HFI--argA.png|600px|thumb|center|The structure of the plasmid pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA ]] |
==Experimental Setup== | ==Experimental Setup== | ||
*Genetic design principle of argA fbr was described on the page of [[Part:BBa_K3595082]] | *Genetic design principle of argA fbr was described on the page of [[Part:BBa_K3595082]] | ||
| Line 18: | Line 18: | ||
==Results== | ==Results== | ||
*The DH10b with plasmid pTYT-argAfbr and ∆argR showed the most significant increase in conversion of ammonia, 40.47% more conversion rate compared to the wildtype | *The DH10b with plasmid pTYT-argAfbr and ∆argR showed the most significant increase in conversion of ammonia, 40.47% more conversion rate compared to the wildtype | ||
| − | + | [[File:T--GZ_HFI--NH3.png|600px|thumb|center|Detection results of the effect of the ammonia pathway. (A) The least-squared regression line of NH4+. (B)The concentration of NH4+. (C) The percentage of decrease in NH4+ compared with M9 medium. ]] | |
<!-- --> | <!-- --> | ||
Revision as of 17:00, 26 October 2020
argA-Wildtype
The ammonia that enters into E.coli is converted to glutamate along with α-Ketoglutarate . The series of reactions that convert glutamate into arginine are catalyzed by N-acetyl glutamate synthetase (NAGS), encoded by gene argA, to acetylize glutamate
Usage and Biology
This part can be used as a coding sequence after the promoter pTac and RBS B0034. The argA protein can be translated under the induction of IPTG. We constructed plasmids pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA using argA and argAfbr, respectively. The constructed plasmid was transformed into E.coli DH10b-ΔargR host cell to test its ammonia degradation efficiency.
Experimental Setup
- Genetic design principle of argA fbr was described on the page of Part:BBa_K3595082
- Plasmid pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA ^ fbr was transfered into the E.coli DH10b -ΔargR host cell,respestively. Meanwhile,plasmid pBR322-KanR-pTac-argA was transfered into the E.coli DH10b.
- Single colonies were selected from the experimental LB-agar plate , then inoculated into test-tube tubes with 4000 μL LB liquid medium with 4uL kanamycin for overnight growth at 37 °C and 200 rpm.
- Inoculate 15 uL of culture solution overnight into a 24-well plate containing 3 mL M9 medium with 3 ul kanamycin with 1.5 uL 1M IPTG for overnight growth at 37 °C and 200 rpm.
- Detecting ammonia concentration in culture medium
Results
- The DH10b with plasmid pTYT-argAfbr and ∆argR showed the most significant increase in conversion of ammonia, 40.47% more conversion rate compared to the wildtype
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 626