Difference between revisions of "Part:BBa K3888999"
 
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<partinfo>BBa_K3888999 short</partinfo>
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<partinfo>BBa_K3888999 short</partinfo><br>
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===description===
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HupS Knockout
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Uptake hydrogenase is a hydrogenase that recycles hydrogen produced by nitrogenase and converts it back into protons, whose expression reduces hydrogen production. The uptake hydrogenase is composed of small and large subunits, encoded by hupS and hupL, respectively. HupS mediates electron transport from the active site of the large subunit to redox partners and downstream reactions through a set of FeS clusters. HupL harbors the active site that contains the metals Ni and Fe.
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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.
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In our experiment, Hupsdownstream flanking region is a part of DNA fragment about 1 KB in length. <br>
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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
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[[File:Deletion Plasmid construction.png]]
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[[File:T--SJTang--Hups_knockout2.png]]
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===Figure 1: Illustration  of the general form of plasmid used to create marked knockout strains of R. palustris. ===
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[[File:T--SJTang--design3.png]]
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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>
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[[File:T--SJTang--hydrogenase-1.png]]
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According to this model, we constructed UppE knockout plasmid below.<br>
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[[ File:HupS Deletion Plasmid Map.png]]
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===Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above.===
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===experiment&results===
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1.We prepared
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2×Taq Mix 25μL,
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2μl HupS Downstream Flanking Region-F,
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2μl HupS Downstream Flanking Region-R ,
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template 1μL(Hups deletion plasmid),  
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ddH2O 20μL
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Same thing is done on upstream flanking region
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to get a total of 50μL PCR, four identical groups are established to make sure accuracy.
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2. During the PCR process, the initial deraturation should be setted at 98 ℃, and it lasts for 3 minutes. The denaturation should be set at 98 ℃ and last for 10s. The temperature of annealing should be 55℃-65 ℃ and it lasts for 30s. The temperature of extention process should be set at 72 ℃ and lasts for 1 minute, final extention should be set at 72 ℃ and lasts for 2 minutes. The temperature for holding is 12 ℃.  
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3. Once it finished, we started to check the purity and concentration of the samples, then do AGE. Here is the result:
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[[File:T--SJTang--Hups_deletion_resulta.png]]
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[[File:T--SJTang--result22.png]]
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4. Electroporation:
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1. Mix 5μL DNA solution(50ng/μL) with competent cells.
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2. Transfer the liquid gained in step 1 into a cuvette.
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3. Set the pulser as 2.0kV, 800Ω, 25μF
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4. Instantly add 1μL SOC medium that has been incubated in "Preparation of Competent Cell".
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5. Shake the mixture on ice for 30 minutes.
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6. Give the mixture a water bath at 37°C for 60 minutes.
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7. Transfer the mixture of bacteria onto a agar plates of YPMOPS.
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Latest revision as of 19:54, 20 October 2021


HupS knockout
HupS Knockout 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.
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.
According to this model, we constructed UppE knockout plasmid below.
HupS Deletion Plasmid Map.png

Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1521
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1521
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1521
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1521
    Illegal NgoMIV site found at 121
    Illegal NgoMIV site found at 258
    Illegal NgoMIV site found at 690
    Illegal NgoMIV site found at 1068
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 414