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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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 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, Hups upstream flanking region is a part of DNA fragment about 1 KB in length for the deletion.
 
In our experiment, Hups upstream flanking region is a part of DNA fragment about 1 KB in length for the deletion.
 
[[File:T--SJTang--Hups_knockout2.png]]
 
[[File:T--SJTang--Hups_knockout2.png]]
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[[File:T--SJTang--design3.png]]
 
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[[File:T--SJTang--hydrogenase-1.png]]
 
  
  
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===experiments&results===
 
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We prepared
 
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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 Upstream Flanking Region
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 19:09, 20 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 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, Hups upstream flanking region is a part of DNA fragment about 1 KB in length for the deletion. T--SJTang--Hups knockout2.png




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