Download BACTERIA TRANSFORMATION LAB (ACTIVITY)

BACTERIA TRANSFORMATION LAB (ACTIVITY)
In this exercise you will use paper to simulate the cloning of a gene from one organism into a
bacterial plasmid using a restriction enzyme digest. The plasmid (puc18 plasmid) can then be
used to transform bacteria so that it now expresses a new gene and produces a new protein.
These diagrams are on the last page of this activity to be easily removed and manipulated.
1. From the colored, cut out the plasmid DNA in a long strip.
2. Attach the ends together to make a loop to simulate the circular DNA of a plasmid.
3. From the white paper, cut out the Mammalian DNA in a long strip. Leave it as a straight
strip. (This is a gene from a vertebrate not a bacterium, so it is not circular.)
The start and stop sequences for transcribing the insulin gene are highlighted.
These are needed to transcribe the gene properly when it is read. In addition, you must find the
restriction enzymes HindIII & EcoR1 cutting sites (sequences of bases)
The two restriction enzymes and their respective restriction sites are listed
below. These enzymes act as “molecular scissors” to cut the DNA at these sequences in the
DNA:
RESTRICTION ENZYME
HindIII
RECOGNITION SITE 5’  3’
A AGCT T
T TCGA A
EcoRI
G AATT C
C TTAA G
4. Notice that the cut plasmid and cut insulin gene now have sticky ends and will match
up perfectly.
5. Tape the insulin gene to the plasmid.
6. You have now made recombinant DNA!!!!
Background:
One of the ways that bacteria remain genetically diverse is through the naturally occurring processes of
transformation. During transformation bacteria take up plasmid DNA from their environment. Plasmids
are small, circular pieces DNA that can be exchanged naturally between bacteria. Plasmids may contain
genes, and when these genes are expressed they can provide bacteria with special traits such as
antibiotic resistance.
Molecular biologists have developed procedures to take advantage of the naturally occurring
transformation process to produce cells that contain desired segments of DNA. Genetic engineering
refers to manipulation techniques used by scientists to change the genetic makeup of an organism. The
basic transformation process is to first select the desired gene to be inserted into the organism and select
a bacterial plasmid, and then cut these two DNA molecules into fragments using special enzymes called
restriction enzymes. The DNA fragments are spliced together with an enzyme called ligase. Finally the
engineered plasmid is taken up by a bacterial cell for replication and expression of the inserted gene.
Enzymes play an important role in the formation of recombinant DNA. To excise the desired gene from
the donor DNA, restriction enzymes are sued to cut DNA bonds in specific locations called recognition
sites. There are many different restriction enzymes and each recognizes and cuts at a different short
sequence of DNA. For example, the restriction enzyme Eco R1 will recognize and cleave DNA at any
section of DNA the reads GAATTC. When the restriction enzyme cuts the DNA, it makes staggered cuts.
This cut results in the production of “sticky ends” that are open to new bonds. By cutting the donor DNA
containing the desired gene and the plasmid DNA with the same restriction enzyme, matching sticky ends
will be produced. The two types of DNA with sticky ends can be joined using another enzyme, DNA
ligase. The newly formed DNA is called recombinant DNA.
In the laboratory, plasmids to be used as molecular carriers or vectors to move specified segments of
DNA into bacteria cells. The bacterial cells will then treat this new DNA as their own and produce the
proteins coded for by the newly introduced segment of DNA.
One of the best examples of transformation success is the production of insulin. The human insulin gene
is isolated and cut from its location on the human chromosome using a restriction enzyme. A plasmid is
cut using the same restriction enzyme. The desired DNA (insulin gene) and plasmid DNA can be joined
using DNA ligase. The plasmid now contains the genetic instructions on how to produce the protein
insulin and contains a gene for antibiotic resistance. By placing bacteria in an appropriate environment,
they can be artificially induced to take up the recombinant DNA plasmids and be transformed.
Eukaryotic cells can also be transformed; however, the process is not quite as simple since these
organisms unlike prokaryotes are frequently multicellular, and the DNA is wound around histone proteins.
In addition eukaryotic genes have introns or intervening DNA sequences that must be removed prior to
translation and the DNA of prokaryotic genes do not contain introns. Some eukaryotic cells that have
been used for genetic engineering are yeast cells which are single celled organisms. Several methods
that have worked in transforming eukaryotic cells are stimulating the cell tot take up DNA using electrical
shock, bombarding the cells with DNA coated projectiles, or injecting the DNA into the zygote cell.
Additionally, viruses can be modified and used to carry desired DNA into a eukaryotic cell.
In this exercise, you will simulate the process of forming a recombinant plasmid using paper models. The
gene of interest is insulin.
Partner 1 NAME _______________________________________________________ DATE _____
Parner 2 NAME ______________________________________________________PERIOD _____
PART I – MODEL BACTERIA TRANSFORMATION
DATA and OBSERVATIONS
Staple recombinant model to both partners handout.
OVERVIEW OF BACTERIA TRANSFORMATION
Directions: Using the box with word choices fill in the numbered boxes with the steps of bacteria
transformation and the letters with the name of the structure next to it.
Word Choices for Letters
foreign DNA with desired
gene
plasmid
recombinant DNA
Word Choices for Numbers
Bacteria transformed with
recombinant plasmid
Plasmid cut with
restriction enzyme
DNA ligase joins sticky
ends to form recombinant
plasmid
Conclusion Questions
1. Describe the role of restriction enzymes in the process of transformation.
2. The restriction enzyme BamH1 cuts DNA between the two G’s when it encounters the base
sequence
GGATCC
CCTAG G
How many recognition sites are found in the segment of DNA below for the restriction enzyme
BamH1? ____________ mark them on the strand
TACGGATCCTAGGGCATAGCTCAGGATCCCGTCAATGGGGATCCC
ATGCCTAGGATCCCGTATCGAGTCCTAGG GCAGTTACCCCTAGGG
3. Why are eukaryotes more difficult to transform than prokaryotes?
PART II – DESIGN A RECOMBINANT CREATURE
Directions: Select one of the desired genes below to create your organism. Record the information in the
spaces and draw your new recombinant organism. Be creative.
A. A gene that causes cells to glow in the dark
B. A gene that codes for the production of a growth hormone
C. A gene that causes the production of glycerol in the cell resulting in freeze resistant cells.
D. A gene that increases the production of muscle proteins.
E. A gene that increases intelligence.
Gene Inserted ____________________________________________________________________
Type of Original Organism Transformed ___________________________________________________
Potential Use of the Recombinant Organism________________________________________________
Sketch of recombinant organism
Plasmid
Plasmid
Chromosomal DNA with
insulin
gene marked
Chromosomal DNA with
insulin
gene marked