Genetic Engineering Notes

taxmanstrongBiotechnology

Dec 11, 2012 (4 years and 9 months ago)

157 views

Unit 3: Genetic Engineering

1
Genetic Engineering: The ability to alter the genetic makeup of an organism by cutting DNA
from one organism and placing it into another in order for the gene to be replicated or
expressed. (Also called recombinant DNA technology)

Steps of Genetic Engineering

1. Obtain the desired gene
2. Insert the desired gene into a host cell
3. Select the cells with the desired gene
4. Induce the host cell to express the desired “foreign” gene
5. Collect and purify the final product

Obtaining the Desired Gene

1. The goal is to find and isolate the section of DNA we are looking for
2. This usually involves isolating the mRNA instead of the DNA
a. mRNA is usually more abundant than the gene itself
b. They contain the same genetic information
c. mRNA is often locally abundant
d. mRNA can be separated from other nucleic acids because of the poly A tail
e. mRNA does not contain introns
3. Once mRNA is isolated, an RNA-DNA hybrid is formed
a. Uses the enzyme reverse transcriptase
b. Synthesizes a complimentary DNA strand using the mRNA as a template
4. mRNA is degraded leaving a single stranded DNA
a. Called cDNA
b. Stands for copy or complimentary DNA
5. cDNA is converted to double stranded DNA using the enzyme DNA polymerase

Inserting the Desired Gene into the Host Cell

1. Inserting fragments of DNA into a bacterial cell so they can direct the production of
proteins is nearly impossible
a. DNA does not easily enter a cell
b. Bacterial cells do not tolerate linear DNA, it must be circular
i. Linear DNA is destroyed by the cell
ii. Genes made from cDNA are linear, not circular
c. DNA in a bacterial cell does not necessarily contain the proper signals for
transcription, translation, and replication
2. Genes must be carried by DNA molecules that occur naturally within bacteria
a. Carriers are called vectors and proper vector selection is critical
b. The most common vector is a plasmid
i. Circular pieces of DNA that replicate independently of the
chromosomal DNA within microorganisms
ii. Why are plasmids valuable as vectors?
Unit 3: Genetic Engineering

2
1. They are small and easily manipulated (usually only 1 or 2
genes)
2. They contain genes for antibiotic resistance
3. They are easily isolated and transferred into cells
4. They can replicate independently
c. How are genes inserted into vectors?
i. Uses specific enzymes called restriction enzymes
1. Cut double stranded DNA through a process called restriction
digestion
2. Each restriction enzyme cuts a specific DNA sequence (over 300
identified)
3. The bases are cut in a staggered fashion creating “sticky ends”
a. Leaves short sections of single strands for
complimentary pairing
4. When complimentary regions come together, DNA ligase
connects the phosphate and the sugar of adjacent nucleotides to
reform the backbone
3. Specific Steps
a. Isolate many copies of the plasmid from bacterial cells
b. Digest the plasmid with a restriction enzyme (creates a linear plasmid with
sticky ends)
c. Attach linkers to the previously isolated DNA fragment
i. These include the correct recognition sequence for the restriction
enzyme being used
d. Digest the desired DNA with the same restriction enzyme (creates sticky ends
that match the plasmid)
e. Mix the digested plasmid and DNA
i. Complimentary sticky ends will join
ii. Fragments are connected using DNA ligase
iii. Possible outcomes
1. Plasmid reforms
2. 2 plasmids join
3. Several genes join
4. Gene and plasmid combination (with or without the desired
gene)
a. The gene is called the insert
b. The plasmid plus the gene is called the construct
f. Transformation
i. Inserting constructs into host cell
ii. Calcium chloride (CaCl
2
) solution causes bacterial cells to take up
plasmids

Selecting the Cells With the Desired Gene

1. There are three issues that must be addressed regarding which cells contain the desired
gene
Unit 3: Genetic Engineering

3
a. Not all cells pick up a plasmid during transformation
b. Not all plasmids contain an insert
c. Not all inserts are the desired gene
2. How can we determine which cells have a plasmid and which cells do not?
a. Antibiotic resistance is the key
b. If we use a plasmid that has a gene for antibiotic resistance, we can separate
cells with the plasmid form cells without
i. All cells are placed onto a plate containing nutrients and an antibiotic
(often ampicillin)
ii. Cells with the plasmid will be resistant and form colonies
iii. Cells without the plasmid will die
3. How can we determine which plasmids have an insert and which do not?
a. Where the plasmid is cut is the key
b. Often uses a technique called blue-white screening
i. We can use a plasmid that contains the gene for -galactosidase, an
enzyme that breaks down lactose
ii. -galactosidase also acts on a chemical called X-gal which is clear
before being cut by -galactosidase and dark blue after
iii. Restriction enzymes can cut within the gene for -galactosidase
iv. If the plasmid has an insert, the -galactosidase will be inactivated
v. If the plasmid does not have an insert, the -galactosidase will be active
1. All cells are placed on a plate containing nutrients, an
antibiotic, and X-gal
2. Cells with no plasmid will die
3. Cells with a plasmid and an insert will be in white colonies
4. Cells with the plasmid and NO insert will be in blue colonies


Inducing the Host Cell to Express the Desired Gene

1. There are three issues that must be addresses regarding the expression of the desired
gene by the host cell
2. The first two are due to the fact that the host cell is prokaryotic while the desired gene
is eukaryotic
a. The genes may contain introns which cannot be removed by prokaryotic cells
b. The genes may contain eukaryotic control signals which can not be used by the
prokaryotic cell
3. The third is due to the fact that the desired gene is foreign to the host cell
a. The product may be degraded or destroyed by the host cell
4. How can we overcome the fact that prokaryotic cells cannot remove introns?
a. The key is using mRNA to obtain the desired gene
i. Eukaryotic cells remove the introns during the production of mRNA
ii. Therefore, the DNA obtained from the mRNA does not contain introns
iii. This is the only known way to solve this problem
5. How can we overcome the fact that prokaryotic cells cannot produce proteins without
prokaryotic control signals?
Unit 3: Genetic Engineering

4
a. The key is using a vector that already contains the appropriate prokaryotic
control signals for transcription and translation and inserting the insert into the
correct location
b. These are called expression vectors and they must be chosen from the
beginning
6. How can we overcome the act that cells destroy foreign molecules?
a. The key is using bacterial cells that are engineered to be lacking proteases
i. Proteases are enzymes that break down foreign proteins
ii. Bacterial cells can be engineered to be unable to produce proteases and
therefore be unable to destroy the desired protein

Collecting and Purifying the Product

1. Once we have the desired product, we remove it from the host cell and must then
separate it from all the other molecules in the cell
2. To do this, we must exploit the differences in the different molecules in the cell
a. Size
b. Charge
c. Solubility
3. Chromatography is used to separate molecules by size
a. Size-exclusion chromatography
i. Uses specially designed polymer beads with very small holes
ii. Molecules of different sizes are forced through a column containing the
beads
iii. Large molecules pass through quickly because they do not interact with
the holes and they are removed from the column
iv. Small molecules come out later because of their interactions with the
holes in the beads