BACTERIAL TRANSFORMATION AP PRE-LAB

Due Friday, Feb 10thor Thursday, Feb 9thby 3:30 for +5 pts!

  • Counts as ½ Summative Grade
  • Pre-lab Quiz on Block, Feb 1st or 2nd= Formative Grade
  • Read Carolina Lab provided AND read Investigation 8 (S97) in your AP lab manual
  • In your lab book:

Page 1

Title

Pre-lab questions – answer questions 1-7 on S-5 & S-6 in Carolina Lab Manual (complete sentences) – Must be completed by Block Day, Feb 1st or 2nd

Leave rest of this page and the next page blank for Introduction and Materials & Methods

Page 3

Construct the Data Tables shown below

Describe the Purpose of each plate and the Expected results. Indicate a prediction of growth with a + and a prediction of growth with a -  Due Friday

  • The terminology is a little confusing. To clarify . . .

LB + → With plasmid, no antibiotics

LB - → No plasmid, no antiobiotics

LB / Amp + → With plasmid and ampicillin

LB / Amp - → No plasmid, with ampicillin

LB / Kan + → With plasmid and kanamycin

LB / Kan - → No plasmid, with kanamycin

  • You must identify your plasmid based on your results. The possibilities are…

pKAN→ Resistance to kanamycin

pAMP→ Resistance to ampicillin

pGREEN→ Green fluorescence + resistance to ampicillin

…………………………………………………………………………………………

Data Analysis:

  • Plasmid Used: ______

Set-Up / Purpose / Expected / Observed
# of Colonies / Description
LB +
LB –
LB/Amp –
LB/Kan –
LB/Amp + / XXX
LB/Kan + / XXX

*Please Note: Data tables must be constructed by Block Day! Be sure to leave enough room for a detailed explanation of the purpose of each plate*

BACTERIAL TRANSFORMATION AP LAB WRITE-UP GUIDELINES

Due Friday, Feb 10th or Thursday, Feb 9th by 3:30 for +5 pts!

Title

  1. Introduction

General overview

Describe how bacteria reproduce, including advantages & disadvantages

Explain the importance and mechanisms for gene transfer in bacteria

Describe the significance of plasmids

Summarize the purpose of the lab

  1. Materials and Methods

Write as a paragraph in past tense … no first person

Summarize procedure followed

For a complete listing of materials and methods, see lab protocol posted by Carolina Biological and College Board, revised by Ms. Lynam.

  1. Data

Data Tables drawn using a ruler & filled in thoroughly

  1. Data Analysis

Calculate the Transformation Efficiency in Part B using instructions found in College Board AP Lab Manual on pp. 106-108 for each plasmid used. Please note: Plasmid used = 10 µL x 0.005 µg/µL

  • Please show your work and record all answers in scientific notation.
  • If you did not have any transformed colonies, use data from another table. Indicate in your write-up which data was used, and provide a possible explanation in your conclusion.

Total Mass of pAMP =

Total Volume =

Fraction Spread =

Mass of pAMP Spread =

Transformation Efficiency =

  1. Questions – Answer all questions except question1 in complete sentences that re-state the question.
  1. If transformation is successful, the antibiotic resistance in the E. coli used in this lab is due to the production of enzymatic protein, which is “built” according to the information encoded in a gene located on the plasmid.

Sketch and label the basic structure of an amino acid monomer.

Illustrate a dipeptide formed by showing two amino acids joined together. In addition, provide the following information …

  1. What type of bond is formed?
  2. What type of reaction occurs?
  3. What waste product is formed?
  4. Specifically, where is this protein construction occurring in the E. coli?
  1. Explain the terms, primary, secondary, tertiary, and quarternary level of protein structure.
  2. Streptomycin and Kanamycin both belong to the same class of antibiotics based on mechanism of action while ampicillin belongs to a different class. Describe the mechanism of action for each class and why it is effective in treating bacterial infections without being toxic to humans.
  3. Assume an E. coli in your gut is busily synthesizing tryptophan, an essential amino acid for building proteins. You ingest a meal containing a large supply of tryptophan, which is now accessible to the E. coli. Provide a detailed explanation of the bacterium’s response and the mechanism this response represents.
  4. Now assume that same E. coli normally metabolizes the glucose you so generously provide; however, glucose supplies have been mostly depleted. Luckily for your gut E. coli, you drink a large glass of milk. Explain in detail the response that will be seen in the bacterium.
  5. Now assume you have provided your gut E. coli with both glucose and lactose. Explain the mechanism that allows the E. coli o preferentially metabolize the glucose first.
  1. Conclusion

General reflection, including whether purpose of the lab was accomplished

Explain whether transformation was accomplished, identification of each plasmid, and how the genetic make-up of each plasmid was determined by the results

Address the calculated Transformation Efficiency values, including the variability between tables using the same plasmid and the variability between different plasmids.

If the procedure was not successful, discuss possible reasons.

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