Difference between revisions of "Part:BBa K3332097"

Latest revision as of 22:30, 27 October 2020

J23100-B0034-phnJ-B0015-J23100-RBS-phnE1-RBS-phnE2

Subunits of phosphonate ABC transporter, permease protein phnE, from S.meliloti 1021. We use BBa_ 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.

Usage

We ligased the J23100-B0034-phnJ-B0015 (BBa_K3332068) 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

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 1057
Illegal NheI site found at 2583
Illegal NheI site found at 2606
Illegal NotI site found at 2750
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 3234
Illegal NgoMIV site found at 3256
Illegal AgeI site found at 2209
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 3246

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3332097 short</partinfo>
-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.
+Subunits of phosphonate ABC transporter, permease protein phnE, from ''S.meliloti'' 1021. We use BBa_ K823004 to construct a new part that can transport glyphosate to cytoplasm.
-<!-- Add more about the biology of this part here
-===Usage and Biology===
+===Biology===
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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===
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We ligased the J23100-B0034-phnJ-B0015 (<partinfo>BBa_K3332068</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===
+'''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>
 <!-- -->