Part:BBa_K3888008
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.
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:
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.
references https://journals.asm.org/doi/10.1128/JB.01248-13
HupS Downstream Flanking Region
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 520
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 520
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 520
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 520
Illegal NgoMIV site found at 67 - 1000COMPATIBLE WITH RFC[1000]
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