Team:Hawaii/Antibiotic test for BB-pRL1383a

From 2008.igem.org

(Difference between revisions)
(→Discussion)
(→Discussion)
Line 137: Line 137:
sp<sub>100</sub> 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. Higher antibiotic concentration also appears to increase the likelihood of obtaining transformants with the desired plasmid+insert.
sp<sub>100</sub> 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. 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 whereas tiny colonies at lower sp concentration were blue. DH5&alpha; supports blue/white selection, so does it mean that our strain has been contaminated with some ''E. coli'' that are not sp resistant but are ''lacZ'' <sup>+</sup>?
 +
:* Will restreak a few tiny blue ones on LB+X-gal to see if they grow. Then colony PCR to verify insert present/absent.

Revision as of 23:47, 23 October 2008

Contents

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:

  • 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

  1. 100 μl of competent DH5α cells were thawed on ice for 10 minutes.
  2. 5 μl of a 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+spvariable. 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+sp40, 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 VF2 primer
    • 1 μl 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

Results

Colony Count of J33207+pRL1383a Transformants
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 + but not sp resistant.

After 19 additional hours, colonies did not turn blue.

A colony PCR of transformants yielded the following gel picture:

File:102308colonypcr.jpg
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.

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. 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 whereas tiny colonies at lower sp concentration were blue. DH5α supports blue/white selection, so does it mean that our strain has been contaminated with some E. coli that are not sp resistant but are lacZ +?

  • Will restreak a few tiny blue ones on LB+X-gal to see if they grow. Then colony PCR to verify insert present/absent.
Retrieved from "http://2008.igem.org/Team:Hawaii/Antibiotic_test_for_BB-pRL1383a"