Difference between revisions of "Part:BBa K3595001"
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<!-- Add more about the biology of this part here --> | <!-- Add more about the biology of this part here --> | ||
=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</i> 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. |
==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]] | ||
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*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. | *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 | *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 | ||
| + | |||
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Revision as of 14:39, 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