Part:BBa_K3888007

HupS Upstream 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, Hups upstream flanking region is a part of DNA fragment about 1 KB in length for the deletion.

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 Upstream Flanking Region

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