Difference between revisions of "Part:BBa K3888008"
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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.
 
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-BBa_3888007-1.png]]
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[[File:T--SJTang-deletion_plasmid-2.png]]
  
 
===experiments&results===
 
===experiments&results===

Revision as of 10:25, 19 October 2021


HupS Downstream Flanking Region

description

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. T--SJTang-deletion plasmid-2.png

experiments&results

We prepared 2×Taq Mix 25μL, 2μl HupS Downstream Flanking Region-F, 2μl HupS Downstream Flanking Region-R , template 1μL(Hups deletion plasmid), ddH2O 20μL To get a total of 50μL PCR, four identical groups are established to make sure accuracy. After PCR, here is the AGE results:image Electroporation: 1. Mix 5μL DNA solution(50ng/μL) with competent cells. 2. Transfer the liquid gained in step 1 into a cuvette. 3. Set the pulser as 2.0kV, 800Ω, 25μF 4. Instantly add 1μL SOC medium that has been incubated in "Preparation of Competent Cell". 5. Shake the mixture on ice for 30 minutes. 6. Give the mixture a water bath at 37°C for 60 minutes. 7. Transfer the mixture of bacteria onto a agar plates of YPMOPS. image


references https://journals.asm.org/doi/10.1128/JB.01248-13


HupS Downstream Flanking Region

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]