Difference between revisions of "Part:BBa K3888007"
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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, 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- | + | [[File:T--SJTang--Hups_knockout2.png]] |
| + | [[File:T--SJTang--design3.png]] | ||
[[File:T--SJTang--hydrogenase-1.png]] | [[File:T--SJTang--hydrogenase-1.png]] | ||
| Line 17: | Line 18: | ||
ddH2O 20μL | ddH2O 20μL | ||
To get a total of 50μL PCR, four identical groups are established to make sure accuracy. | To get a total of 50μL PCR, four identical groups are established to make sure accuracy. | ||
| − | After PCR, here is the AGE results: | + | After PCR, here is the AGE results: |
| + | [[File:T--SJTang--Hups_deletion_resulta.png]] | ||
| + | [[File:T--SJTang--result22.png]] | ||
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| + | |||
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Electroporation: | Electroporation: | ||
1. Mix 5μL DNA solution(50ng/μL) with competent cells. | 1. Mix 5μL DNA solution(50ng/μL) with competent cells. | ||
| Line 26: | Line 33: | ||
6. Give the mixture a water bath at 37°C for 60 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. | 7. Transfer the mixture of bacteria onto a agar plates of YPMOPS. | ||
| − | + | ||
Revision as of 14:43, 20 October 2021
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:
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 Upstream Flanking Region
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 121
Illegal NgoMIV site found at 258
Illegal NgoMIV site found at 690 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 414