Difference between revisions of "Part:BBa K3332024"
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===Usage=== | ===Usage=== | ||
| − | We ligased the strong | + | We ligased the strong promoter、RBS、Terminator(<partinfo>BBa_J23100</partinfo>、<partinfo>BBa_B0034</partinfo>、<partinfo>BBa_B0015</partinfo>) and the parts (phnJ)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=== | ||
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After receiving the synthesized DNA, digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1. | 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| | + | <table><tr><th>[[File:T--XMU-China2020--BBa K3332024.png|thumb|720px|Fig.1 The result of plasmid cut with enzyme ''Eco''RI and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table> |
'''2. Enzyme activity''' | '''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. | + | |
| + | 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). | ||
<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> | <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> | ||
| − | + | ===Sequence and Features=== | |
| − | + | ||
<partinfo>BBa_K3332024 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3332024 SequenceAndFeatures</partinfo> | ||
Latest revision as of 01:25, 28 October 2020
phnJ
Subunit of C-P lyase, which can degrade the glyphosate, from Enterobacterales. This subunit is essential to the activity of the whole enzyme.Use BBa_K823004 to construct a new part that can improve the capability of chassis bacteria to degrade glyphosate.
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 strong promoter、RBS、Terminator(BBa_J23100、BBa_B0034、BBa_B0015) and the parts (phnJ)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
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.
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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 113
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 597
Illegal NgoMIV site found at 619 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 609