"
User:University of Washington/1 August 2008
From 2008.igem.org
LuxR from AraC and TetR
- BBa_P1010 (contains death gene) grew in both XL1-Blue and DB3.1 which was weird. XL1-Blue didn't have death resistance, so the cell shouldn't grow. Will miniprep and sequence the part P1010. But will use the vector plasmid containing Amp resistance gene from the part nevertheless.
- Obtained P1010 on pSB3K3 and pSB1AC3 from Brandi by inoculated the strain on selective plates.
LuxR from pLac
-E0240 insert gel piece and R0010 vector gel piece were used with a gel purification kit to extract the DNA from them.
-Isolated DNA samples were nanodropped. DNA concentration of both the E0240 insert DNA sample and R0010 vector DNA sample were found to be about 5 ng/ul.
-Vector and insert were ligated together using biocircuits protocol.
Cure MG1655Z1
- Restriction digested the Elowitz's plasmid from original plate and stock plate with XbaI and BamHI (both cut both plasmid pACYC184 and pCD26 once)
| Reagents | Ori + XbaI | Stock + XbaI | Ori + BamHI | Stock + BamHI | Control |
|---|---|---|---|---|---|
| dH2O | 34 ul | 34 ul | 39 ul | 39 ul | 39.5 ul |
| Buffer | 5 ul NEB2 | 5 ul NEB2 | 5 ul NEB3 | 5 ul NEB3 | 5 ul NEB3 |
| BSA | 0.5 ul | 0.5 ul | 0.5 ul | 0.5 ul | 0.5 ul |
| DNA | 10 ul | 10 ul | 5 ul | 5 ul | 5 ul |
| vortex | |||||
| Enzyme | 0.5 ul XbaI | 0.5 ul XbaI | 0.5 ul BamHI | 0.5 ul BamHI | - |
| centrifuge and incubate 37 degree Celsius | |||||
- After 2:45 hours incubation, 30 ul of cut DNA was used to run on gel. All, except control, showed two bands: one between 4-5 kb, the other between 8-10 kb. (Expected pCD26 9.5 kb, pACYC184 4.2 kb) Possible conclusion was the strain contained plasmid pACYC184 that produce LuxR as mentioned in the literature.
- 8 colonies were selected from second Tsy plate and inoculated on LB + Kan plate to check if we had lost the promoter plasmid(pCD26 with KAN resistance).
Back to Team:University_of_Washington/Notebook#Notebook
