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.<br> | In our experiment, Hups upstream flanking region is a part of DNA fragment about 1 KB in length for the deletion.<br> | ||
| + | [[File:Deletion Plasmid construction.png]] | ||
| + | ===Figure 1: Illustration of the general form of plasmid used to create marked knockout strains of R. palustris. === | ||
| + | The origin of replication (ori), origin of transfer (oriT) and sacB (blue) are part of the delivery plasmid. The green sequence is inserted into the multiple cloning site of the delivery plasmid. The left and right fragments (green) are sequences amplified from the R. palustris genome and used in overlap extension PCR to generate a recombinant product containing (a deletion and a restriction site）. The restriction site is used to introduce the resistance cassette (purple). This interrupts the gene even if no deletion is present, and allows selection of successful transformants. <br> | ||
| + | According to this model, we constructed UppE knockout plasmid below.<br> | ||
[[File:T--SJTang--Hups_knockout2.png]] | [[File:T--SJTang--Hups_knockout2.png]] | ||
| − | + | ===Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above.=== | |
| − | + | ||
Revision as of 19:12, 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.
Figure 1: Illustration of the general form of plasmid used to create marked knockout strains of R. palustris.
The origin of replication (ori), origin of transfer (oriT) and sacB (blue) are part of the delivery plasmid. The green sequence is inserted into the multiple cloning site of the delivery plasmid. The left and right fragments (green) are sequences amplified from the R. palustris genome and used in overlap extension PCR to generate a recombinant product containing (a deletion and a restriction site）. The restriction site is used to introduce the resistance cassette (purple). This interrupts the gene even if no deletion is present, and allows selection of successful transformants.
According to this model, we constructed UppE knockout plasmid below.
Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above.
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