Team:Hawaii/Antibiotic test for BB-pRL1383a

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#:* Oops. 100 &mu;l was plated on LB+sp<sub.100</sub> plate.
#:* Oops. 100 &mu;l was plated on LB+sp<sub.100</sub> plate.
# Plates were incubated at 37C for 19 hours.
# Plates were incubated at 37C for 19 hours.
 +
===Verification of plasmid construct===
 +
# Plates were stored at 4C for 19 hours after incubating at 37C.
 +
# 6 colonies from LB+sp<sub>40, 50, 75, 100</sub> 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
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#:* Final elongation: 72C for 10 minutes
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#:* Cycles: 30
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#:<br>
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#:* PCR reaction:
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#::* 1 &mu;l [http://partsregistry.org/wiki/index.php?title=Part:BBa_G00100 VF2 primer]
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#::* 1 &mu;l [http://partsregistry.org/wiki/index.php?title=Part:BBa_G00101 VR primer]
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#::* 3 &mu;l PCR water
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#::* 5 &mu;l REDTaq DNA Polymerase mixture (Sigma-Aldrich)
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#::* colony (a sterile toothpick was used to scrape part of the colony to inoculate the PCR reaction)
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# 2 &mu;l PCR product were loaded onto a 1% EtBr stained 2% agarose gel for visualization
==Results==
==Results==

Revision as of 23:55, 22 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 [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+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 [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

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+ 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.

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