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Team:Hawaii/Antibiotic test for BB-pRL1383a
From 2008.igem.org
Contents |
Antibiotic test and Blue/White screen for BB-pRL1383a
Objective
- To verify the successful construction of BB-pRL1383a, a pRL1383a derivative in BioBrick format with blue/white screening capabilities
- To determine the ideal concentration of spectinomycin for isolation of E. coli colonies transformed with BB-pRL1383a
- To verify the ability to use blue/white screening for selection of transformed cells and to determine length of incubation necessary for this screen
Materials and Methods
Media
To test for antibiotic selection, 30 ml LB + 1.5% agar plates were made. Spectinomycin were added at the following concentrations:
- sp2.5
- sp5
- sp10
- sp15
- sp20
- sp25
- sp40
- sp50
- sp75
- sp100
Thirty microliters of 20X X-gal (20 mg/ml) was added to each plate to a final concentration of 1 μg/ml.
Control plates, also 30 ml LB + 1.5% agar, were as follows:
- LB only
- + X-gal (1 μg/ml)
- + sp100
Transformation
- 100 μl of competent DH5α cells were thawed on ice for 10 minutes.
- 5 μl of a J33207 and pRL1383a ligation reaction were added to the cells. Ten microliters of cells were left untransformed.
- Cells were incubated on ice for 5 minutes.
- Cells were heat shocked at 42C for 1 minute, then returned to ice for 2 minutes.
- 400 μl SOC were added to the transformed cell mixture and 50 μl were added to the untransformed aliquot.
- Cells were incubated at 37C with shaking at 250 rpm for 70 minutes.
- 50 μl of transformed cells + SOC were plated LB+spvariable. 20 μl of untransformed cells + SOC were plated on control plates.
- Oops. 100 μl was plated on LB+sp<sub.100</sub> plate.
- Plates were incubated at 37C for 19 hours.
Results
| Plate | Cells | Colonies | Blue? |
|---|---|---|---|
| LB (+) control | untransformed DH5α | lawn | no |
| LB+X-gal (+) control | untransformed DH5α | lawn | yes |
| LB+sp100 (-) control | untransformed DH5α | 0 | no |
| LB+sp2.5 | J33207+pRL1383a ligation in DH5α | lawn | yes |
| LB+sp5 | J33207+pRL1383a ligation in DH5α | lawn | yes |
| LB+sp10 | J33207+pRL1383a ligation in DH5α | lawn | yes |
| LB+sp15 | J33207+pRL1383a ligation in DH5α | lawn | yes |
| LB+sp20 | J33207+pRL1383a ligation in DH5α | lawn | yes |
| LB+sp25 | J33207+pRL1383a ligation in DH5α | lawn | yes |
| LB+sp40 | J33207+pRL1383a ligation in DH5α | 56 large, too many to count pinpoint, smear present | no |
| LB+sp50 | J33207+pRL1383a ligation in DH5α | 29 large, too many to count pinpoint, smear present | no |
| LB+sp75 | J33207+pRL1383a ligation in DH5α | 41 large, ~30 pinpoint | no |
| LB+sp100 | J33207+pRL1383a ligation in DH5α | 75 large, 12 small | no |
After 19 hours, distinct colonies could be seen on plates with sp concentration >/= 40. However, none of these colonies were blue, suggesting that sp may somehow interfere with expression of lacZ. The controls show that the transformation process did not kill the E. coli and that DH5α are lacZ+</sub> but not sp resistant.
Discussion
Antibiotic selection is necessary to distinguish between the colonies that contain BB-pRL1383a and those that do not.
sp100 appears to be the lowest concentration of antibiotic necessary for single colony isolation. Lower concentrations resulted in growth of untransformed E. coli. The smear created by these bacteria gradually faded as antibiotic concentration was increased.
