Difference between revisions of "Part:BBa K3888007"
 
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<partinfo>BBa_K3888007 short</partinfo>
 
<partinfo>BBa_K3888007 short</partinfo>
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===description===
 
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The HupS gene encodes a partial uptake hydrogenase subunit. Mutating the hupS gene inactivated the uptake hydrogenase enzyme, thus increasing hydrogen production. The experimental plan is to obtain a 1.3 KB DNA fragment containing hupS gene and about 0.5 KB upstream and downstream flanking region DNA fragment by PCR, and clone them into pJQ200SK plasmid. The engineered plasmid will then be transferred by e. coli S17-1 conjugation or electroporation into R. Palustris CGA009.
 
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In our experiment, Hups upstream flanking region is a part of DNA fragment about 1 KB in length for the deletion.
 
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[[File:T--SJTang-BBa_3888007-1.png]]
 
  
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===experiments&results===
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HupS Upstream Flanking region<br>
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We prepared
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The HupS gene encodes a partial uptake hydrogenase subunit. Mutating the hupS gene inactivated the uptake hydrogenase enzyme, thus increasing hydrogen production. The experimental plan is to obtain two 1KB upstream and downstream flanking region DNA fragment by PCR, and gibson with pK18mobsacB-Gm plasmid backbone.Then the plasmid was imported into R. palustris via elctrotransformation.
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2×Taq Mix 25μL,
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In our experiment, Hups upstream flanking region is a part of DNA fragment about 1 KB in length for the deletion.<br>
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2μl HupS Downstream Flanking Region-F,
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[[File:Deletion Plasmid construction.png]]
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2μl HupS Downstream Flanking Region-R ,
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===Figure 1: Illustration  of the general form of plasmid used to create marked knockout strains of R. palustris. ===
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template 1μL(Hups deletion plasmid),
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The origin of replication (ori), origin of transfer (oriT) and sacB (blue) are part of the delivery plasmid. The green sequence is inserted into the multiple cloning site of the delivery plasmid. The left and right fragments (green) are sequences amplified from the R. palustris genome and used in overlap extension PCR to generate a recombinant product containing (a deletion and a restriction site）. The restriction site is used to introduce the resistance cassette (purple). This interrupts the gene even if no deletion is present, and allows selection of successful transformants. <br>
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ddH2O 20μL
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According to this model, we constructed UppE knockout plasmid below.<br>
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To get a total of 50μL PCR, four identical groups are established to make sure accuracy.
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[[ File:HupS Deletion Plasmid Map.png]]
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After PCR, here is the AGE results:image
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===Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above.===
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Electroporation:
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1. Mix 5μL DNA solution(50ng/μL) with competent cells.
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2. Transfer the liquid gained in step 1 into a cuvette.  
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3. Set the pulser as 2.0kV, 800Ω, 25μF
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4. Instantly add 1μL SOC medium that has been incubated in "Preparation of Competent Cell".
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5. Shake the mixture on ice for 30 minutes.
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6. Give the mixture a water bath at 37°C for 60 minutes.
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7. Transfer the mixture of bacteria onto a agar plates of YPMOPS.
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image
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references
 
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https://journals.asm.org/doi/10.1128/JB.01248-13
 
  
  
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HupS Upstream Flanking Region
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:43, 21 October 2021


HupS Upstream Flanking Region

HupS Upstream Flanking region
The HupS gene encodes a partial uptake hydrogenase subunit. Mutating the hupS gene inactivated the uptake hydrogenase enzyme, thus increasing hydrogen production. The experimental plan is to obtain two 1KB upstream and downstream flanking region DNA fragment by PCR, and gibson with pK18mobsacB-Gm plasmid backbone.Then the plasmid was imported into R. palustris via elctrotransformation. In our experiment, Hups upstream flanking region is a part of DNA fragment about 1 KB in length for the deletion.
Deletion Plasmid construction.png

Figure 1: Illustration of the general form of plasmid used to create marked knockout strains of R. palustris.

The origin of replication (ori), origin of transfer (oriT) and sacB (blue) are part of the delivery plasmid. The green sequence is inserted into the multiple cloning site of the delivery plasmid. The left and right fragments (green) are sequences amplified from the R. palustris genome and used in overlap extension PCR to generate a recombinant product containing (a deletion and a restriction site）. The restriction site is used to introduce the resistance cassette (purple). This interrupts the gene even if no deletion is present, and allows selection of successful transformants.
According to this model, we constructed UppE knockout plasmid below.
HupS Deletion Plasmid Map.png

Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 121
    Illegal NgoMIV site found at 258
    Illegal NgoMIV site found at 690
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 414