From 2008.igem.org

Antibiotic test and Blue/White screen for BB-pRL1383a

Objective

1. To verify the successful construction of BB-pRL1383a, a pRL1383a derivative in BioBrick format with blue/white screening capabilities
2. To determine the ideal concentration of spectinomycin for isolation of E. coli colonies transformed with BB-pRL1383a
3. 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:

• sp_2.5
• 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

1. 100 μl of competent DH5α cells were thawed on ice for 10 minutes.
2. 5 μl of a [http://partsregistry.org/Part:BBa_J33207 J33207] and pRL1383a ligation reaction were added to the cells. Ten microliters of cells were left untransformed.
3. Cells were incubated on ice for 5 minutes.
4. Cells were heat shocked at 42C for 1 minute, then returned to ice for 2 minutes.
5. 400 μl SOC were added to the transformed cell mixture and 50 μl were added to the untransformed aliquot.
6. Cells were incubated at 37C with shaking at 250 rpm for 70 minutes.
7. 50 μl of transformed cells + SOC were plated LB+sp_variable. 20 μl of untransformed cells + SOC were plated on control plates.

   • Oops. 100 μl was plated on LB+sp<sub.100</sub> plate.

8. Plates were incubated at 37C for 19 hours.

Verification of plasmid construct

1. Plates were stored at 4C for 19 hours after incubating at 37C.
2. 6 colonies from LB+sp_{40, 50, 75, 100} plates were colony PCR'd to verify presence of insert (and by inference, plasmid)

   • Initial denaturing: 94C for 10 minutes
   • Denature: 94C for 30 seconds
   • Anneal: 62C for 30 seconds
   • Elongate: 72C for 75 seconds
   • Final elongation: 72C for 10 minutes
   • Cycles: 30

   
   

   • PCR reaction:

   • 1 μl [http://partsregistry.org/wiki/index.php?title=Part:BBa_G00100 VF2 primer]
   • 1 μl [http://partsregistry.org/wiki/index.php?title=Part:BBa_G00101 VR primer]
   • 3 μl PCR water
   • 5 μl REDTaq DNA Polymerase mixture (Sigma-Aldrich)
   • colony (a sterile toothpick was used to scrape part of the colony to inoculate the PCR reaction)

3. 2 μl PCR product were loaded onto a 1% EtBr stained 2% agarose gel for visualization
4. Colonies #1, 2 from sp₁₀₀ plate and a (tiny) blue colony from sp_2.5 plate were restreaked on LB (+), LB+amp₁₀₀ (to verify J33207 plasmid transformation from before wasn't a fluke due to contamination), and LB+sp₁₀₀ (purification). X-gal was added at the aforementioned concentration to all plates. Plates were incubated at 37C for 13 hours.

Verification of results

1. The entire experiment was repeated using DH5α MCR cells obtained from Douglas Risser from SC lab.

Results

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 ⁺ but not sp resistant.

After 19 additional hours, colonies did not turn blue.

Antibiotic test plates, 38 hours after initial plating.

A colony PCR of transformants yielded the following gel picture:

EtBr stained 2% agarose gel ran at 95V for 1 hour. Two microliters of PCR product were loaded into each well.

Two colonies from the sp₄₀ plate appeared to have the correct insert. None of the colonies PCR'd contained the correct insert from the sp₅₀ plate. All PCR'd colonies from the sp₇₅ and sp₁₀₀ plates appeared to have the desired insert.

Restreaked DH5α. Row 1: Resteak of blue colony from sp_2.5 plate. Row 2: Restreak of colonies from sp₁₀₀ plate. L->R: LB+Amp₁₀₀+X-gal, LB+X-gal, LB+sp₁₀₀+X-gal.

Observations were collected 19 hours after initial plating. The "contamination" bacteria (blue) are not ampicillin resistant. Colonies that expressed the original J33207 (lac promoter + lacZ) as supplied in pSB1A2 by iGEM were true transformants and not products of contaminated DH5α competent cells.

The experiment was repeated using DH5α MCR cells obtained from Douglas Risser of the SC lab to verify ideal antibiotic concentration.

BBa_J33207 transformed into DH5α MCR and plated on LB+Amp₁₀₀. (+) control.

After 17 hours, distinct colonies could be seen on plates with sp concentration >/= 40. The controls show that the transformation process did not kill the E. coli, DH5α are not sp resistant, and J33207 (in pSB1A2) is a functional part. It is troubling that none of the colonies transformed with our plasmid are blue.
A colony PCR of transformants yielded the following gel picture:

EtBr stained 2% agarose gel ran at 95V for 1 hour. Three microliters of PCR product were loaded into each well.

Control plates. Untransformed DH5α MCR plated on (L->R): LB+Xgal, LB+sp_2.5+X-gal, LB+sp₁₀₀+X-gal.

Test plates. J33207+pRL1383a transformed DH5α MCR plated on LB+sp_variable plates (Top->Bottom, L->R): sp_2.5, sp₅, sp₁₀, sp₂₀, sp₂₅, sp₄₀, sp₅₀, sp₇₅, sp₁₀₀.

Discussion

Antibiotic selection is necessary to distinguish between the colonies that contain BB-pRL1383a and those that do not.

sp₁₀₀ appears to be the ideal concentration of antibiotic necessary for single colony isolation, though single colonies can be observed at sp₄₀. Lower concentrations resulted in growth of untransformed E. coli. The smear created by these bacteria gradually faded as antibiotic concentration was increased. Higher antibiotic concentration also appears to increase the likelihood of obtaining transformants with the desired plasmid+insert.

However, it was observed that none of the colonies at higher sp concentrations were blue. The tiny colonies from the first trial at lower sp concentration were blue. It was likely that our DH5α strain was been contaminated with some E. coli that are not sp resistant but are lacZ ⁺.

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