Difference between revisions of "Part:BBa K3332101"
Line 8: Line 8:
 
Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. ''Enterobacterales'' use phnHIJK genes to encode C-P lyase, PhnJ protein is an essential subunit that can crack C-P bond. The 21th arginine was mutated to methionine.
 
Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. ''Enterobacterales'' use phnHIJK genes to encode C-P lyase, PhnJ protein is an essential subunit that can crack C-P bond. The 21th arginine was mutated to methionine.
 
===Usage===
 
===Usage===
−
We ligased the strong promoter (BBa_J23100) and the parts (RBS-phnJ_mut21-Terminator) on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into ''E. coli'' DH5α & ''E. coli'' BL21(DE3), enabled the ''E. coli'' to degrade glyphosate at higher efficiency and improved the binding ability with the endogenous PhnHIK.
+
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We ligased the J23100-B0034-Mut21_phnJ-B0015 (<partinfo>BBa_K3332069</partinfo>) and the part J23100-B0034-phnE<sub>1</sub>-B0034-phnE<sub>2</sub>(<partinfo>BBa_K3332067</partinfo>) on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into ''E. coli'' DH5α & ''E. coli'' BL21(DE3), enabled the ''E. coli'' to degrade glyphosate at higher efficiency.
 +
 
 
===Characterization===
 
===Characterization===
 
'''Enzyme activity'''
 
'''Enzyme activity'''
  
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We use Negative Control, experiment groups phnEE and Mut21_phnJ-phnEE to verify that Mut21_phnJ can enhance the degradation of glyphosate by the chassis bacteria by our detection system. The Mut21_phnJ-phnEE gene cluster enhances the degration of glyphosate by the chassis bacteria compared to the blank control, but it has no difference with the phnE1E2 group, indicating that exogenous phnJ can’t bind to the endogenous PhnHIK well.  
+
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We use Negative Control, experiment groups phnEE and Mut21_phnJ-phnEE to analyze if Mut21_phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut21_phnJ gene lowers the degradation rate of ''E.coli'' BL21(DE3) about 16.4%, indicate the mutant has higher binding ability to PhnHIK but lower degradation ability compare to endogenous PhnJ.  
  
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The result is shown in Fig.2(Experiment groups in Fig.2  
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The result is shown in Fig.2(Experiment groups in Fig.2  

Revision as of 22:29, 27 October 2020


J23100-RBS-phnJ_mut21-B0015-J23100-RBS-phnE1-RBS-phnE2

Subunits of phosphonate ABC transporter, permease protein phnE, from S.meliloti 1021. We use K823004 to construct a new part that can transport glyphosate to cytoplasm.

Biology

Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. Enterobacterales use phnHIJK genes to encode C-P lyase, PhnJ protein is an essential subunit that can crack C-P bond. The 21th arginine was mutated to methionine.

Usage

        We ligased the J23100-B0034-Mut21_phnJ-B0015 (BBa_K3332069) and the part J23100-B0034-phnE1-B0034-phnE2(BBa_K3332067) on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), enabled the E. coli to degrade glyphosate at higher efficiency.

Characterization

Enzyme activity

        We use Negative Control, experiment groups phnEE and Mut21_phnJ-phnEE to analyze if Mut21_phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut21_phnJ gene lowers the degradation rate of E.coli BL21(DE3) about 16.4%, indicate the mutant has higher binding ability to PhnHIK but lower degradation ability compare to endogenous PhnJ.

        The result is shown in Fig.2(Experiment groups in Fig.2

       Negative Control: J23100-B0034_pSB1C3

       phnE1E2: J23100-B0034-phnE1-B0034-phnE2_pSB1C3,

       phnJ-phnE1E2: J23100-B0034-phnJ-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3,

       Mut21-phnJ-phnE1E2: J23100-B0034-phnJ_mutR21M-B0015- J23100-B0034-phnE1-B0034-phnE2_pSB1C3

       Mut1640-phnJ-phnE1E2: J23100-B0034-phnJ_mutT16S&R40Y-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3

       RNAi system-phnJ-phnO-phnE1E2: J23101-OmpA 5'UTR-phnF 0.97-Hfq binding sequence-J23101- OmpA 5'UTR-phnJ 0.69-Hfq binding sequence- BBa_J61048-J23100-B0034-phnJ-B0015-J23100-phnO-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3).

Fig.2 Relationship between concentration of glyphosate and culture time.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 1057
    Illegal NheI site found at 2583
    Illegal NheI site found at 2606
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3186
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3234
    Illegal AgeI site found at 2209
    Illegal AgeI site found at 2905
    Illegal AgeI site found at 3412
  • 1000
    COMPATIBLE WITH RFC[1000]