E-Analysis & Assessment Name:
AP Lab: Bacterial Transformation with Luciferase
Respond to the questions below. Click in the space under the question so that your text is 14pt leaving the question text 12pt. If graphing is required, create it in Microsoft Excel and paste it into this document. Do not print! Upon completion, save this file, adding your name before the file name (e.g. Joe Shmoe - LuciferaseTransformation.doc). Attach and email to Mr. Z (). Note: In addition to the background information in the lab handout, you may need to consult your textbook, class notes, or search the Internet.
Analysis
1.Use your cellphone or camera to photograph your results. Paste the image below with a descriptive caption.
2.Transformation efficiency is the number of resistant colonies per microgram of plasmid. Use the directions below to calculate transformation efficiency. Show all work! (Note: The tube of plasmid pBestluc supplied in this activity contains 1 ug of plasmid in 100 uL of buffer.)
a) Number of colonies on plate =
Total mass of plasmid used
b) Concentration of plasmid (in ug/uL) =
b) Volume of plasmid solution used (from procedure) =
c) Total mass of plasmid used (volume x concentration) =
Fraction of transformation suspension placed on plate
d) Total volume of suspension (uL CaCl + uL plasmid + uL Luria broth) =
e) Fraction placed on plate (volume placed on plate/total volume of suspension) =
Total mass of plasmid on plate
f) Total mass of plasmid used x fraction of suspension placed on plate =
Number of colonies per ug of plasmid
g) Number of colonies on plate / Total mass of plasmid on plate =
Assessment
3.Based on your observations, did transformation occur? EXPLAIN why or why not?
4.Explain why the Luria agar plate you used contained the antibiotic ampicillin.
- Suppose you performed a transformation and obtained the results on the three plates shown below. You added 10 uL of plasmid at a concentration of 0.005 ug/uL to cells suspended in 300 uL of CaCl2. After your heat-shock step, 200 uL of Luria broth was added to each reaction. After the experiment was complete, you placed 200 uL of the final solution on each plate and incubated. Calculate the transformation efficiency of each reaction and then calculate the average transformation efficiency for all 3 trials. Show work!
Total mass of plasmid used =
Total volume of suspension =
Fraction of suspension on plate =
Total mass of plasmid in fraction =
Number of colonies per ug of plasmid
Plate 1 =
Plate 2 =
Plate 3 =
Avg. =
- For whichever of the following statements are NOT true, explain why under that statement.
a) Cells can only be transformed when they are in a competent state.
b) Transformation may only be performed using plasmid DNA containing antibiotic-resistant genes (R factors).
c) Transformed cells are capable of passing their newly-acquired traits onto succeeding generations.
d) Transformation was first discovered in the bacterium Streptococcus pneumoniae.
e) Cells must first be treated artificially in the laboratory before they are capable of undergoing transformation.
- Chose from the following list of terms to complete the paragraphs below: (Note: Be sure the words you insert remain highlighted.)
Alexander Flemingliving non-pathogenictransforming factor1940s
conjugationOswald AverytransductionHershey & Chase
heat-killed pathogenicliving pathogenic1928Hydrolytic
RNA1895rabbits Frederick Griffith
transformationheat-killed non-pathogenicWatson & Crick guinea pigs
T.H. MorganStreptococcus pneumoniaeBacillus subtilis Eschericia coli
miceDNAprotein 1953
The phenomenon of was first discovered in by . In his now-famous experiment, he injected with cells of the bacterium and with cells of the same bacterium. The results displayed the properties of cells. This led him to conclude that the cells must have been altered in some way by the material from the cells. Though the exact substance causing the change in the cells was unknown at the time, he proposed that some sort of caused the genetic exchange when the two types of cells were combined.
Years later, in the early , the work of , along with several colleagues, demonstrated exactly what the substance was that transformed the cells. By using enzymes to isolate specific components from the cells and exposing each component individually to the cells, it was demonstrated the only material that could cause transformation of the cells was . Their work was met with much skepticism initially because until that point it had been assumed that was the source of genetic material. However, repetition of the experiment and the work of proved conclusively that is indeed the transforming substance, as well as the source of genetic material.
8.Transformation is one type of genetic exchange among bacteria. Research another type of genetic exchange that occurs in bacteria. Either draw a fully labeled diagram showing the mechanism (scan & insert below), or Google the best diagram you can find (copy & paste below). Either way, include a brief descriptive caption.
9.What are some possible benefits of recombinant DNA technology and genetic engineering? What are some potential drawbacks?