Difference between revisions of "Part:BBa K5291037"
Kortybones (Talk | contribs) (→Usage and Biology) |
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We use this system to prevent plasmid loss and conduct bacteria suicide.<br><br> | We use this system to prevent plasmid loss and conduct bacteria suicide.<br><br> | ||
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<br><b>Fig.1 The schema of the <i>hok</i>/<i>sok</i> system.</b><br><br> | <br><b>Fig.1 The schema of the <i>hok</i>/<i>sok</i> system.</b><br><br> | ||
And we successfully construct the strain with plasmid pAB-<i>hok</i>/<i>sok</i>. <br><br> | And we successfully construct the strain with plasmid pAB-<i>hok</i>/<i>sok</i>. <br><br> | ||
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<br><b>Fig.2 The AGE result of pAB1-<i>hok</i>/<i>sok</i>.</b><br><br> | <br><b>Fig.2 The AGE result of pAB1-<i>hok</i>/<i>sok</i>.</b><br><br> | ||
To verify the function of plasmid anti-loss, we take advantage of the strains with and without <i>hok</i>/<i>sok</i> system, cultivating them in non-resistent LB broth to deliberately induce plasmid loss. After that we transfer them to the non-resistent plates and then select single colonies, scribing them on resistance plates to examine if they still own the plasmid we introduce. We find that after several generation passing the death rate of the groups without <i>hok</i>/<i>sok</i> system is significantly higher than the one with the system, which means that the system is beneficial to plasmid maintaining.<br><br> | To verify the function of plasmid anti-loss, we take advantage of the strains with and without <i>hok</i>/<i>sok</i> system, cultivating them in non-resistent LB broth to deliberately induce plasmid loss. After that we transfer them to the non-resistent plates and then select single colonies, scribing them on resistance plates to examine if they still own the plasmid we introduce. We find that after several generation passing the death rate of the groups without <i>hok</i>/<i>sok</i> system is significantly higher than the one with the system, which means that the system is beneficial to plasmid maintaining.<br><br> | ||
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<b>Fig.3 The 17th LB-Amp plate of the verification of plasmid anti-loss function. (Left: <i>hok</i>/<i>sok</i>+ Right: <i>hok</i>/<i>sok</i>-)</b><br><br> | <b>Fig.3 The 17th LB-Amp plate of the verification of plasmid anti-loss function. (Left: <i>hok</i>/<i>sok</i>+ Right: <i>hok</i>/<i>sok</i>-)</b><br><br> | ||
<html><img width = "550" src="https://static.igem.wiki/teams/5291/images/part-cr/hoksok-diagram.png" /></html><br> | <html><img width = "550" src="https://static.igem.wiki/teams/5291/images/part-cr/hoksok-diagram.png" /></html><br> | ||
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We verify the plasmid anti-loss function of <i>hok</i>/<i>sok</i> system.<br><br> | We verify the plasmid anti-loss function of <i>hok</i>/<i>sok</i> system.<br><br> | ||
We also conduct the experiment to examine the function of cell suicide when there is no activator of P<sub>opdH</sub>. Since citrate, the main activator of P<sub>opdH</sub>, will be taken up by <i>Pseudomonas aeruginosa</i> as the carbon source, we add another promoter <i>pLac</i> which can be activated by IPTG, a kind of substance stable in cells, in front of <i>sok</i> just for experimental research. We devide the bacteria with pAB-<i>hok</i>/<i>sok</i> into two groups, cultivating in the LB-Amp broth with and without IPTG, and testing OD600 of each group after cultivation.<br><br> | We also conduct the experiment to examine the function of cell suicide when there is no activator of P<sub>opdH</sub>. Since citrate, the main activator of P<sub>opdH</sub>, will be taken up by <i>Pseudomonas aeruginosa</i> as the carbon source, we add another promoter <i>pLac</i> which can be activated by IPTG, a kind of substance stable in cells, in front of <i>sok</i> just for experimental research. We devide the bacteria with pAB-<i>hok</i>/<i>sok</i> into two groups, cultivating in the LB-Amp broth with and without IPTG, and testing OD600 of each group after cultivation.<br><br> | ||
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<html><img width = "500" src="https://static.igem.wiki/teams/5291/images/part-cr/od600-hoksoktest2.png" /></html><br> | <html><img width = "500" src="https://static.igem.wiki/teams/5291/images/part-cr/od600-hoksoktest2.png" /></html><br> | ||
| − | <b>Fig. | + | <b>Fig.5 The contrast between the groups with and without IPTG and the diagram of the different OD600 of the two groups.</b><br><br> |
We can know from the diagram that the OD600 of the group with IPTG is much higher than the other group, which means the better growth of the former group. We successfully verify the specific control of the <i>hok</i>/<i>sok</i> system. | We can know from the diagram that the OD600 of the group with IPTG is much higher than the other group, which means the better growth of the former group. We successfully verify the specific control of the <i>hok</i>/<i>sok</i> system. | ||
<br>*The concentration of IPTG we choose is 0.6mM, which is found as the best concentration of induction in our pre-laboratory.<br><br> | <br>*The concentration of IPTG we choose is 0.6mM, which is found as the best concentration of induction in our pre-laboratory.<br><br> | ||
Latest revision as of 13:04, 2 October 2024
pAB-hok/sok
We construct the hok/sok system to realize plasmid anti-loss as well as bacteria suicide when they are out of the mangroves.
Usage and Biology
We use this system to prevent plasmid loss and conduct bacteria suicide.
Fig.1 The schema of the hok/sok system.
And we successfully construct the strain with plasmid pAB-hok/sok.
Fig.2 The AGE result of pAB1-hok/sok.
To verify the function of plasmid anti-loss, we take advantage of the strains with and without hok/sok system, cultivating them in non-resistent LB broth to deliberately induce plasmid loss. After that we transfer them to the non-resistent plates and then select single colonies, scribing them on resistance plates to examine if they still own the plasmid we introduce. We find that after several generation passing the death rate of the groups without hok/sok system is significantly higher than the one with the system, which means that the system is beneficial to plasmid maintaining.
Fig.3 The 17th LB-Amp plate of the verification of plasmid anti-loss function. (Left: hok/sok+ Right: hok/sok-)
Fig.4 The diagram shown the death rate of bacteria in two groups.
We verify the plasmid anti-loss function of hok/sok system.
We also conduct the experiment to examine the function of cell suicide when there is no activator of PopdH. Since citrate, the main activator of PopdH, will be taken up by Pseudomonas aeruginosa as the carbon source, we add another promoter pLac which can be activated by IPTG, a kind of substance stable in cells, in front of sok just for experimental research. We devide the bacteria with pAB-hok/sok into two groups, cultivating in the LB-Amp broth with and without IPTG, and testing OD600 of each group after cultivation.
Fig.5 The contrast between the groups with and without IPTG and the diagram of the different OD600 of the two groups.
We can know from the diagram that the OD600 of the group with IPTG is much higher than the other group, which means the better growth of the former group. We successfully verify the specific control of the hok/sok system.
*The concentration of IPTG we choose is 0.6mM, which is found as the best concentration of induction in our pre-laboratory.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 146
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 16
- 1000COMPATIBLE WITH RFC[1000]