Difference between revisions of "Part:BBa K3332100"

Revision as of 19:05, 27 October 2020

RNAi sequence of phnF-RNAi sequence of phnJ-J23100-RBS-phnJ-T-J23100-RBS-phnO-T-J23100-RBS-phnE1E2

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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.

Usage

We ligased the RNAi system (J23101-OmpA 5'UTR-phnF 0.97-Hfq binding sequence-J23101- OmpA 5'UTR-phnJ 0.69-Hfq binding sequence- BBa_J610480) and the phn system (RBS-phnJ-Terminator-RBS-phnO-Terminator-RBS-phnE1-RBS-phnE2) 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.

Characterization

1. Agarose Gel Electrophoresis

After receiving the synthesized DNA, digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1.

Fig.1 The result of plasmid cut with enzyme EcoRI and PstI. Plasmid: pSB1C3.

2. Enzyme activity

We use Negative Control, experiment groups phnEE and phnEEJ to verify that phnJ can enhance the degradation of glyphosate by the chassis bacteria by our detection system. The phnJ-phnE1E2 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.

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 438
Illegal NheI site found at 461
Illegal NheI site found at 869
Illegal NheI site found at 892
Illegal NheI site found at 1921
Illegal NotI site found at 1036
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2134
Illegal XhoI site found at 281
Illegal XhoI site found at 712
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1520
Illegal NgoMIV site found at 1542
Illegal AgeI site found at 4812
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 1532

@@ Line 5: / Line 5: @@
 None
-<!-- Add more about the biology of this part here
+===Biology===
-===Usage and 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.
+===Usage===
+We ligased the RNAi system (J23101-OmpA 5'UTR-phnF 0.97-Hfq binding sequence-J23101- OmpA 5'UTR-phnJ 0.69-Hfq binding sequence- BBa_J610480) and the phn system (RBS-phnJ-Terminator-RBS-phnO-Terminator-RBS-phnE1-RBS-phnE2) 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.
+===Characterization===
+'''1. Agarose Gel Electrophoresis'''
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After receiving the synthesized DNA, digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1.
+<table><tr><th>[[File:T--XMU-China2020--BBa K3332024.png|thumb|300px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table>
+'''2. Enzyme activity'''
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We use Negative Control, experiment groups phnEE and phnEEJ to verify that phnJ can enhance the degradation of glyphosate by the chassis bacteria by our detection system. The phnJ-phnE1E2 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;The result is shown in Fig.2(Experiment groups in Fig.2
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Negative Control: J23100-B0034_pSB1C3
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;phnE1E2: J23100-B0034-phnE1-B0034-phnE2_pSB1C3,
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;phnJ-phnE1E2: J23100-B0034-phnJ-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3,
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mut21-phnJ-phnE1E2: J23100-B0034-phnJ_mutR21M-B0015- J23100-B0034-phnE1-B0034-phnE2_pSB1C3
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mut1640-phnJ-phnE1E2: J23100-B0034-phnJ_mutT16S&R40Y-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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).
+<table><tr><th>[[File:T--XMU-China2020--BBa K3332024 3.png|thumb|500px|Fig.2 Relationship between concentration of glyphosate and culture time.]]</th><th></table>
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