Difference between revisions of "Part:BBa K3332066"
| Line 13: | Line 13: | ||
After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1. | After receiving the synthesized DNA, restriction 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 K3332021.png|thumb|300px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3..]]</th><th></table> | <table><tr><th>[[File:T--XMU-China2020--BBa K3332021.png|thumb|300px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3..]]</th><th></table> | ||
| − | 2. HPLC | + | 2. Agarose Gel Electrophoresis |
| + | After receiving the synthesized DNA, restriction 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 K3332067 1.png|thumb|300px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table> | ||
| + | 3. HPLC | ||
Verify that phnEE enhances the transport of glyphosate by the chassis bacteria by HPLC. The result is shown in fig.2, phnEE enhance the transport of glyphosate by the chassis bacteria compared to the blank control obviously. | Verify that phnEE enhances the transport of glyphosate by the chassis bacteria by HPLC. The result is shown in fig.2, phnEE enhance the transport of glyphosate by the chassis bacteria compared to the blank control obviously. | ||
<table><tr><th>[[File:T--XMU-China2020--BBa K3332067 2.png|thumb|300px|Fig.2 Relationship between elution peak area of glyphosate and culture time]]</th><th></table> | <table><tr><th>[[File:T--XMU-China2020--BBa K3332067 2.png|thumb|300px|Fig.2 Relationship between elution peak area of glyphosate and culture time]]</th><th></table> | ||
| − | |||
<!-- --> | <!-- --> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 06:57, 25 October 2020
J23100-RBS-phnE2
Subunit of phosphonate ABC transporter, permease protein phnE, from S.meliloti 1021.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. Sinorhizobium meliloti 1021 use PhnE1 and PhnE2 to construct the permease protein of ABC transporter, which includes PhnE1, PhnE2, PhnC and PhnD proteins, this transporter can transport glyphosate to cytoplasm. The phnE2 gene encodes a subunit of permease protein which can transport glyphosate to cytoplasm.
Usage
We ligased the strong promoter (BBa_J23100) and the parts (RBS-phnE2-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 express PhnE2 protein.
Characterization
1. Agarose Gel Electrophoresis After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1.
2. Agarose Gel Electrophoresis After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1.
3. HPLC Verify that phnEE enhances the transport of glyphosate by the chassis bacteria by HPLC. The result is shown in fig.2, phnEE enhance the transport of glyphosate by the chassis bacteria compared to the blank control obviously.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 68 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1220
- 1000COMPATIBLE WITH RFC[1000]