Difference between revisions of "Part:BBa K3888008"
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<partinfo>BBa_K3888008 short</partinfo>
 
<partinfo>BBa_K3888008 short</partinfo>
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===description===
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HupS Downstream 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 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.
 
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.
 
In our experiment, Hupsdownstream flanking region is a part of DNA fragment about 1 KB in length.  
 
In our experiment, Hupsdownstream flanking region is a part of DNA fragment about 1 KB in length.  
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[[File:T--SJTang--Hups_knockout2.png]]
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[[File:Deletion Plasmid construction.png]]
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[[File:T--SJTang--design3.png]]
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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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[[File:T--SJTang--hydrogenase-1.png]]
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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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According to this model, we constructed UppE knockout plasmid below.<br>
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===experiments&results===
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[[ File:HupS Deletion Plasmid Map.png]]
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We prepared
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===Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above.===
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2×Taq Mix 25μL,
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2μl HupS Downstream Flanking Region-F,
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2μl HupS Downstream Flanking Region-R ,
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template 1μL(Hups deletion plasmid),  
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ddH2O 20μL
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To get a total of 50μL PCR, four identical groups are established to make sure accuracy.
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After PCR, here is the AGE results:
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[[File:T--SJTang--Hups_deletion_resulta.png]]
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[[File:T--SJTang--result22.png]]
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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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references
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https://journals.asm.org/doi/10.1128/JB.01248-13
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HupS Downstream Flanking Region
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 19:38, 20 October 2021


HupS Downstream Flanking Region HupS Downstream 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 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. In our experiment, Hupsdownstream flanking region is a part of DNA fragment about 1 KB in length. 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
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 520
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 520
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 520
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 520
    Illegal NgoMIV site found at 67
  • 1000
    COMPATIBLE WITH RFC[1000]