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Team:NTNU Trondheim/Alternative systems
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==Alternative system== | ==Alternative system== | ||
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| + | [[File:pVB20system.png|thumb|Plasmid map of system in pVB20wt.]] | ||
From the results, promoter rrnB P1 is not transcribing enough repressor to inhibit pLac-promoter. To test out if the other parts of the system work (LacI-pLac-mCherry), a test system where Pm-promoter controls expression of LacI was made. The wild-type Pm-promoter that was used here is induced by m-toluate. | From the results, promoter rrnB P1 is not transcribing enough repressor to inhibit pLac-promoter. To test out if the other parts of the system work (LacI-pLac-mCherry), a test system where Pm-promoter controls expression of LacI was made. The wild-type Pm-promoter that was used here is induced by m-toluate. | ||
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| - | LacI+LacP+MC in plasmid pUC128 was isolated.To retrieve the LacI-pLac-mCherry insert pUC128 was cut with EcoRI and the fragments were separated on gel. The insert fragment were cut out and extracted from the gel. | + | LacI+LacP+MC in plasmid pUC128 was isolated.To retrieve the LacI-pLac-mCherry insert pUC128 was cut with EcoRI and the fragments were separated on gel. The insert fragment were cut out and extracted from the gel as shown in Figure 1. |
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| - | To transfer it to pVB20wt, pVB20wt was opened using EcoRI and then treated with CIP to avoid religation, and was then ligated to the EcoRI | + | To transfer it to pVB20wt, pVB20wt was opened using EcoRI and then treated with CIP to avoid religation, and was then ligated to the EcoRI-digested insert fragment. |
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| + | The final plasmid is shown in Figure 1. | ||
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| + | {{:Team:NTNU_Trondheim/NTNU_footer}} | ||
Latest revision as of 10:52, 31 August 2011
Alternative system
From the results, promoter rrnB P1 is not transcribing enough repressor to inhibit pLac-promoter. To test out if the other parts of the system work (LacI-pLac-mCherry), a test system where Pm-promoter controls expression of LacI was made. The wild-type Pm-promoter that was used here is induced by m-toluate.
LacI+LacP+MC in plasmid pUC128 was isolated.To retrieve the LacI-pLac-mCherry insert pUC128 was cut with EcoRI and the fragments were separated on gel. The insert fragment were cut out and extracted from the gel as shown in Figure 1.
To transfer it to pVB20wt, pVB20wt was opened using EcoRI and then treated with CIP to avoid religation, and was then ligated to the EcoRI-digested insert fragment.
The final plasmid is shown in Figure 1.
