Teacher Notes:

This will take at least 2 days.

Day one:

-Give students directions on how to color the strips, mark the enzymes and tape the strips together.

-After day one they should have this completed

Day two:

-Make the restriction map and the DNA map

-Give directions about which is a good enzyme.

-Demonstrate that they should cut along the selected enzyme cut site and tape to form recombinant plasmid

-Answer questions.

Recombinant DNA Using Bacterial Plasmids

Background

Bacteria have not only their normal DNA, they have a circular DNA also. It is a wonderful ally for biologists who desire to get bacteria to produce very specific proteins. The plasmids conveniently can be cut, fused with other DNA and reabsorbed by bacteria. The bacteria easily incorporates the new DNA information into its metabolism. This “recombining” of DNA is called RECOMBINANT DNA.

There are a number of RESTRICTION ENZYMES that are available to cut the plasmid DNA and the new DNA strands that need to be “pruned” closer to the specific DNA message that will be fused with the plasmid.

Goals

In this activity, a make-believe DNA message for the protein insulin is marked on the cell DNA. Your task will be to find an enzyme that cuts the plasmid once (and only once) and cuts the cell DNA as a close possible on both ends of the insulin code - so that the insulin code can be fused into the circle of the plasmid DNA.

To do this you will need to:

Determine which restriction enzyme to use to cut your DNA segments.

Determine which antibiotic you would use to determine if your finalized recombined DNA was absorbed by the DNA or not. *note: antibiotics are NOT the same as enzymes or insulin, they are used to kill bacteria.

Materials

  • White instructions page
  • White answer sheet
  • Colored (GREEN ) bacterial plasmid
  • Colored (RED ) Cell DNA
  • Colored (BLUE) insulin gene
  • Colored resistance genes and plasmid replication site
  • Scissors
  • Tape

Instructions

  1. READ AND FOLLOW DIRECTIONS CLOSELY.
  1. Obtain scissors and a piece of tape.
  1. Color the plasmid strips GREEN, but do NOT color the antibiotic resistance genes or the plasmid replication sequences. Color the ampicillin resistance gene ORANGE. Color the kanamycin gene PURPLE. Color the Tetracyclin resistance gene BROWN. Color the plasmid replication site YELLOW. Cut the PLASMID strips. Put the four strips in order by matching the letters to make a circular plasmid.
  1. Color the cell DNA strips RED, except the insulin gene color BLUE. Cut out the CELL DNA strips. They must be taped together in the order indicated at the bottom of each strip. Make sure to match the numbers to each other to form one long linear strip. Note where the DNA code for insulin (protein gene) is located.
  1. After completing steps 3 & 4, use the PLASMID MAP to map out the relative locations of the DNA code for each of the antibiotic resistances. Use a pencil to mark the positions of the genes for antibiotic resistance that your plasmid contains. Label each.

YOU ARE NOW READY FOR TO BEGIN ONE OF THE TWO MAJOR PARTS OF THIS ACTIVITY

Part 1

  1. It is time to begin testing the various restriction enzymes that you have. You should have 6 restriction enzymes. These 6 restriction enzymes are given to cut the DNAs. Note that each of the restriction enzyme rectangles, there is the name of the enzyme (such as HindIII) and a short DNA sequence that shows exactly what sequences the enzyme cuts. Using the sequences given find ALL cut site locations on both the cell DNA and the plasmid. When you find a site mark it with a pencil showing the restriction site and labeling which enzyme cuts it.
  1. Once you have all the restriction sites marked and labeled on BOTH the cell DNA and the plasmid you can fill out the chart on the Recombinant DNA using plasmids worksheet. You can also mark the locations of the restriction sites on the back side of the paper for both the plasmid and cell DNA.
  1. Your job as a biochemist is to find a restriction enzyme that will cut open your plasmid at ONE site only. The same enzyme should be able to cut your cell DNA at TWO sites, one above and one below the gene for insulin. It is very important that you find an enzyme that cuts as close to the insulin gene as possible (Without cutting the insulin gene). Based on this information you need to select and justify which enzyme you will use.
  1. After you have completed testing the enzymes, select which ONE enzyme you would use to cut the plasmid and the Cell DNA. Use scissors to make the cut in your plasmid and the Cell DNA. Be certain to make the cuts in the staggered fashion made by the actual enzyme. This will expose the “sticky ends” where joining will be possible. (Since one enzyme was used, all “sticky ends” will be compatible.) Use tape to splice you insulin gene into the plasmid chain. You have now created a RECOMBINANT DNA!!!! 

Part 2

In a real situation, you would mix your recombinant DNA plasmids with the bacteria of your choice. These bacteria would absorb the plasmids out of their environment and act as the hosts. These host bacteria should then begin producing insulin. You could purify the insulin and sell it so that it could be used by diabetics.

Early in this activity, you were asked to note and record which of the antibiotic resistances you had on your plasmid. This knowledge is extremely useful in determining if and in which bacteria took up the plasmid as they were supposed to. The host bacteria are normally killed by the antibiotics (kanamycin, ampicillin, and tetracycline). However, if the recombinant plasmids were actually taken up by the bacteria, the plasmids may have contained on them the DNA gene for resisting the effects of one or more antibiotics. Therefore, if the host bacteria are placed in a growth medium containing an antibiotic to which they have a resistant gene in their recombinant plasmid DNA, they will survive. Any bacteria that failed to take in recombinant plasmids could die in the growth medium. THEREFORE, the host bacteria that survive have taken in the recombinant plasmids, those that do not survive have not taken it in.

  1. Fill in the rest of the appropriate spaces in your table on the answer sheet. Five exact reasons why you did not use the certain enzymes and the reason why you chose the one enzyme you did. Also answer the rest of the questions on the “Recombinant DNA using Bacterial Plasmids worksheet.”
  2. When finished staple the completed recombinant plasmid to your paper to turn it in.

Restriction Enzymes

T A
T A
C G HindIII
G C
A T
A T /
C G
C G
T A BamHI
A T
G C
G C
G C
CG
AT PxnII
C G
GC / C G
T A
T A EcoRI
A T
A T
G C
G C
G C
G C XmaI
C G
C G
C G / T A
T A
C G SacI
A T
A T

Name:______Date: ______Period: _____

Plasmid restriction sitemap

Cell DNA restriction site map

Recombinant DNA using Bacterial Plasmids

Part 1: Restriction Enzyme Data Table

Name of restriction enzyme / Number of cuts on:
Plasmid DNA / Length of DNA fragments / Used
(X) / Not used
(X) / Exact reason for use or nonuse
BamHI
EcoRI
HindIII
HpaII
XmaI
SacI

Part II.

  1. Which antibiotic(s) could you use in your growth medium to test for plasmid uptake?Explain why.
  1. Which antibiotics(s) could NOT be used in your growth medium to test for plasmid uptake. Explain why.
  1. What is the difference between precursor mRNA and functional mRNA? Why is this difference important in recombinant DNA techniques?
  1. What happens if the insulin gene would have been cut in the middle instead of at the end?
  1. Write a brief paragraph outlining what you would do with the recombinant plasmid to insert it into bacteria, and then selectively grow just the bacteria with your insulin gene.

Cell DNA

T A
G C
G C
G C
C G
C G
T A
A T
G C
G C
C G
A T
C G
GC
G C
G C
G C
C G
C G
C G
G C / G C
A T
G C
A T
T A
T A
C G
T A
T A
A T
A T
G C
T A
TA
CG
A T
AT
C G
A T
G C
G C / T A
T A
C G
G C
A T
A T
G C
G C
T A
A T
C G
A T
T A
A T
A T
C G
G C
T A
C G
T A
TA / CG
AT
AT
G C
T A
C G
A T
T A
G C
T A
G C
C G
C G
T A
T A
T A
T A
A T
A T
A T
T A / G C
T A
A T
A T
T A
A T
T A
T A
C G
C G
T A
C G
C G
T A
T A
A T
A T
G C
A T
A T
T A / T A
C G
G C
A T
A T
A T
T A
T A
TA
AT
AT
G C
G C
G C
C G
C G
C G
T A
A T
G C
G C

Bacterial Plasmid

T A
A T
G C
G C
C G
C G
C G
C G
C G
T A
T A
CG
AT
AT
A T
G C
G C
G C
A T
C G
T A / C G
G C
A T
G C
T A
T A
A T
A T
C G
C G
T A
A T
G C
G C
A T
G C
G C
G C
C G
C G
C G / T A
G C
G C
T A
G C
G C
G C
G C
G C
C G
A T
A T
G C
G C
T A
T A
A T
T A
A T
C G
T A / T A
A T
GC
C G
A T
C G
G C
T A
A T
G C
G C
T A
T A
C G
G C
A T
A T
C G
T A
C G
C G

= AMPICILLIN RESISTANCE= KANAMYCIN RESISTANCE

= TETRACYCLIN RESISTANCE= PLASMID REPILCATION