Unit 4Recombinant DNA Technology

Lesson 1 – Recombinant DNAIsolating and Ligating DNA• Lecture – Isolating and Ligating DNA• Activity – Clone a paper plasmid

Recombinant DNA Technology – Isolating and Ligating DNA• A gene must be isolated and well

characterized before it can be used in genetic manipulations.• One method of isolating and

amplifying DNA of interest is to clone the gene by inserting it into a DNA molecule that serves as a vehicle or a vector.•When cells divide, the

recombinant DNA will be reproduced.

Recombinant DNA Technology-Isolating and Ligating DNASteps in gene cloning• 1. Isolation of DNA (gene of

interest) using restriction enzymes.• 2. Ligating selected DNA to a

vector (bacterial plasmid).• 3. Transformation of host cells

with recombinant DNA (Inserting recombinant plasmid into cell)• 4. Selection of host cells with the

recombinant DNA.• 5. Production of an appropriate

proteins.

Recombinant DNA Technology – Isolating and Ligating DNA

• Restriction Enzymes and DNA Plasmids• Restriction enzyme = DNA cutting enzymes• Plasmid = Circular form of self replicating DNA.

Recombinant DNA Technology – Isolating and Ligating DNA• Restriction enzymes are primarily found in bacteria.• They are given abbreviated names based on the genus and

species of the bacteria from which they were isolated.• Ex. EcoRI was isolated from E.coli strain RY13• Restriction enzymes cut DNA by cleaving the sugar-

phosphate backbone.• Restriction enzymes do not randomly cut, nor do they all cut

DNA in the same location.

Recombinant DNA Technology – Isolating and Ligating DNA• Like other enzymes restriction enzymes show specificity for

certain sites.• Restriction enzymes recognize, bind to, and cut DNA within

specific base sequences called restriction sites.

Recombinant DNA Technology – Isolating and Ligating DNA• Restriction enzymes are

called 4 or 6 base cutters because they typically recognize restriction sites with 4 or 6 nucleotides.• Each restriction site is a

palindrome. The nucleotides read the same way backwards and forwards.

Recombinant DNA Technology – Isolating and Ligating DNA• Some restriction enzymes

cut DNA with fragments with overhanging single stranded ends called sticky or cohesive ends.• Other enzymes generate

fragments with double stranded blunt ends.

Recombinant DNA Technology – Isolating and Ligating DNA

Recombinant DNA Technology – Isolating and Ligating DNA

• Biotechnologists prefer sticky ends over blunt end cutters because DNA fragments can be joined easily together.•When DNA from two sources

is joined together, the enzyme DNA ligase is used to catalyze bonding between sugar and phosphate groups in the DNA backbone.

Recombinant DNA Technology – Isolating and Ligating DNA

• DNA from a “foreign” source (plant, animal, viral, bacterial, yeast) is generally bonded to vector DNA. Vectors can be bacterial plasmids (most typical), yeast, viruses, or artificial chromosomes and are used to transfer the recombinant DNA. • After cutting DNA with restriction enzymes , biotechnologists will

sometimes check for molecular size to ensure recombinant DNA procedures have worked.• They will employ gel electrophoresis test to isolated restriction

fragments of interest .• Let’s review what we learned last year:• http://www.dnalc.org/resources/animations/gelelectrophoresis.html• http://learn.genetics.utah.edu/content/labs/gel/

Activity – Clone a paper plasmid• Read the directions on the handout.• Complete the activity and respond to questions.• Submit your completed activity to the teacher.

Lesson 1 – What you need to know

•What are the steps in gene cloning?• Describe how restriction enzymes cut DNA.• Define a restriction site and a palindrome.•What is the difference between sticky and blunt ends?• Explain in detail how the DNA isolation and ligating

procedures are accomplished.

Lesson 2 - Transformation

• Webquest – Bacterial transformation process• Lecture: Selection of transformed bacterial cells

Transformation

• Transformation – is the process of inserting foreign DNA into a bacteria reliably.• The purpose of this technique is to introduce a foreign plasmid into a

bacteria and to use that bacteria to amplify the plasmid with its gene of interest in order to make large quantities of it. • The gene of interest inserted into the bacteria may be a protein such

as insulin or Factor VIII for blood clotting as examples. The bacteria produce large quantities of the protein and it can be sold commercially.

Lesson 2 - Transformation• Lab transformation process 1. Host bacterial cells are treated with calcium chloride solution. 2. Recombinant plasmids are added to bacterial cells and chilled on

ice. 3. Then the cells and DNA mixture are briefly heated.• The recombinant plasmids will enter the bacteria cell, replicate, and

express the genes.

Lesson 2 - Transformation

• http://www.phschool.com/science/biology_place/labbench/lab6/intro.html• Go to the above

website and complete the webquest.• Respond to all

webquest questions.• Class review of

responses.

Transformation • Selection• The transformation process is not perfect because not all bacterial cells

will contain the recombinant plasmid.• A selection process needs to be in place to find the bacterial cells that

have been transformed.• A plasmid containing resistance to an antibiotic (usually ampicillin) is used

as a vector . The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that are sensitive to ampicillin. • The bacteria are then spread over a plate that contains ampicillin. The

ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate. • Therefore, as long as you grow the bacteria in ampicillin, it will need the

plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted to the plasmid.

Transformation

Transformation• Concepts behind GFP

Lab• Review of the lac operon• Parts of operon: - Promoter - Operator - Repressor Protein - Inducer - Genes of Interest

Transformation• If repressor protein is

present alone, it binds to operator.• This prevents RNA

polymerase from attaching to the promoter.• Result: No

transcription of operon gene(s).

Transformation• If an inducer is present,

it binds to repressor protein.• Inducer-repressor

complex cannot bind to operator.• RNA polymerase

attaches to promoter and transcription of the operon gene(s) will follow.

Transformation• In this lab, the vector

plasmid carries a gene for ampicillin resistance (AmpR) and the green fluorescent protein (gfp).• The gfp gene has been

inserted into the lac operon genes.• The vector plasmid is then

inserted into E.coli via the transformation procedure

Transformation• Once the transformation is

complete, cells are plated onto LB agar, LB agar with ampicillin, and LB agar with ampicillin and an inducer called IPTG.• If IPTG is present, it binds to the

lac repressor, and transcription of the gfp protein will occur.• Your next assignment is to carry

out the transformation procedure and determine which bacterial cells have been transformed.

Lesson 2 – What you need to know

• Describe how transformed bacterial cells are located and selected after the transformation procedure.• Explain how the lac operon works.• Describe how the recombinant plasmid with gfp and Amp R

operates.

Lesson 3 – Transformation Lab

• Day 1 – Perform Transformation• Day 2 – Interpret Results

Lesson 4 – Webquest and Debate

• Read Golden Rice Case Study and learn how rice is transformed. Respond to all assigned questions. Class discussion of responses. http://openlearn.open.ac.uk/mod/oucontent/view.php?id=398600• Research the pros and cons of the genetic modification of golden rice

for a debate.• Debate: Golden rice is a genetically modified food that is fortified to

prevent vitamin A deficiency; used particularly in developing nations. The question to be debated , “Is the use of golden rice a good strategy to prevent vitamin A deficiency in developing nations?”• See your handout for debate instructions.