medical-notes/content/Biokemi/Plasmidlabb/Plasmid Lab Part 1 OCR.md

Recombinant DNA technology
Lecture by:
Anne Wöhr
anne.wohr@gu.se

Recombinant DNA technology
Molecular cloning Transformation Selection and 
Replication
Performed during this lab

Plasmids
• Commonly found in bacteria as extra-chromosomal 
circular dsDNA molecules 
• Able to self-replicate during cell division
• Often carry beneficial genes like antibiotic resistance
• Bacteria can share genetic information through 
plasmid transfer
By: Maya Kostman
How could this be useful for us?

Applications of recombinant DNA 
technologies
Khan S., 2016
Biopharmaceuticals:
Vaccines eg hepatitis B vaccine
recombinant proteins eg Insulin (Diabetes), Factor VIII (hemophilia)
Genetically modified organisms: 
Organisms that have been genetically modified to exhibit specific traits eg 
herbicide-resistant crop plants
Gene Therapy: 
In some genetic disorders, patients lack the functional form of a gene. Gene 
therapy attempts to provide a normal copy of the gene to the cells of the 
patient.
Gene Analysis: 
build artificial, recombinant versions of genes that help understand how 
genes in an organism function

• Scissors: DNA restriction enzymes (DNA digestion)
• Cutting sites: multiple cloning sites (MCS)
• Glue: DNA ligase (DNA ligation)
• Host: bacterial cells (Transformation)
• Environment: LB medium or LB agar plates (Culturing)
• Goal: making more identical copies, or expression (making proteins)
Toolbox for molecular cloning

✓ DNA molecule that acts as a vehicle to carry foreign genetic materials 
into another cell, where it can be replicated or expressed.
Vectors
Ori (origin of replication): 
Replication is initiated here, enabling the plasmid to reproduce itself.
MCS (multiple cloning site):
Short segment of DNA which contains many restriction sites. This
allows a piece of DNA to be inserted into that region. The used
plasmid contains a BamHI cleavage site in its MCS.
AmpR gene: 
encodes the enzyme beta-Lactamase, which inactivates ampicillin.
Cells containing a plasmid vector which expresses AmpR can be
selected from those that do not by growth in an ampicillin-containing
medium.
Lac promoter: 
binding site of RNA polymerase to initiate expression. IPTG binds
and inactivates the LacI repressor protein and thereby enables
expression of genes downstream of the promoter.
LacZ⍺ gene: 
encodes the alpha-peptide of the enzyme beta-galactosidase.
Functional beta-galactosidase consits of the alpha- and omega-
peptide. The used E-coli strain carries the lacZ deletion mutant
which contains the omega-peptide but lacks the alpha-peptide. The
activity of mutant beta-galactosidase is rescued by the presence of
the alpha-peptide present in the plasmid (alpha-complementation).

Restriction enzymes (Scissors)
✓ Sequence-specific DNA endonucleases
✓ Recognise and cleave DNA sequences at specific restriction sites
✓ Generate “sticky end” or “blunt end”

Escherichia coli (Host)
✓ Model organism in molecular biology
✓ Gram negative, rod shaped bacteria
✓ Located in lower intestine

The History of Insulin Production
1921: Discovery of insulin
1922: Leonard Thompson became the first person with diabetes ever 
treated through administration of insulin
1923: Insulin is commercialized
Insulin sales kit, Eli Lilly and Company, 1940s
14 cows or 70 pigs to sustain a diabetic 
patient for 1 year
1970: Recombinant DNA technology is 
developed
1982: Recombinant insulin is commercialized 

Production of Insulin
Adapted from “From DNA to Beer: Harnessing Nature in Medicine & Industry”

Purpose of this lab: 
Determine whether a gene of interest has 
been successfully cloned into a vector.

Transformation

Transformation
During the incubation 
on ice, DNA binds to 
the surface of the 
bacterium as a calcium-
phosphate-DNA 
complex
Following a sudden 
increase in 
temperature, one or 
more DNA molecules 
bound to the surface of 
the cell is taken up by 
the competent cell

How can we selectively grow bacteria 
that have taken up the plasmid?

Selection pressure
✓ ampR gene encodes for beta-lactamase
✓ Inactivates ampicilin antibiotics
✓ Only cells containing vector DNA will grow
in the presence of ampicilin
Selection based on antibiotic resistance

How do we select for bacteria with the plasmids 
carrying the inserts?
By: Maya Kostman

More detailed info: thermofisher.com
LacZ gene naturally found in E. coli, encodes β-galactosidase.
We use an E.coli strain that carries the LacZ deletion mutant which
contains the omega-peptide but lacks the alpha-peptide and is therefore
non-functional. The plasmid we use carries the alpha-peptide, rescuing the
function of mutant beta-galactosidase.
Blue-white screening 

Lab Schedule
• Monday (11/24)
Introductory lecture 12:15 – 13:00
Lab 13:15 – 14:00 Groups 1-19
Lab 14:15 – 15:00 Groups 20-38 
• Tuesday (11/25)
Lab 11:15 – 11:45 Groups 1-19 
Lab 12:00 – 12:30 Groups 20-38
• Wednesday (11/26) Groups 1-19 
Introductory lecture 8:15 – 9:00
Lab 09:15 – 16:00
• Thursday (11/27) Groups 20-38
Introductory lecture 8:15 – 9:00
Lab 9:15 – 16:00
Deadline for submitting lab reports: 07/12/2025

Working in the lab
✓ Work in groups of 2 people, stick to your assigned partner 
✓ Always wear gloves and lab coat to protect you and your samples, 
wash your hands thoroughly before leaving the lab
✓ When using the pipette, check the volume limits (0.1-10μl, 10-200μl, 
and 100-1000μl)
✓ Pipette into the bottom of the tube, do not ”shoot” it (especially when
working with very low volumes)

Transformation of competent cells
→ performed during this lab (day 1)
✓ 1 tube of plasmids (P) (with/without insert)
✓ 1 tube of competent bacteria (C)

Day 1: Selection of transformed cells
→ performed during this lab (day 1)
✓ Pick up bacterial colonies (2 white, 2 blue) from plate
✓ Grow in LB medium with antibiotics (expand the colony and replicate plasmids)
Day 2: Picking & expansion of blue and white
colonies
→ performed during this lab (day 2)

Day 1: Materials for Transformation
Falcon tube (50ml) LB-agar plate Eppendorf tube 
Day 1: Spread plate method
o Apply light pressure to not tear or stab the agar

Day 3 - work overview
✓ Purify plasmids 
✓ Restriction enzyme digestion
✓ Run on agarose gel
✓ Interpret results

Lab reports
✓ Write according to the guidelines on the handout on Canvas
✓ One lab report per group (your names and project group number on 
the cover page)
✓ In English
✓ Upload your lab reports on CANVAS deadline on 07/12/2025

Lecture Questions
1. Name three applications of molecular cloning.
2. What is a plasmid?
3. Name the three main steps involved in recombinant DNA technology?
4. What is a restriction enzyme?
5. What are the bacteria used in your protocol?
6. Explain the calcium/phosphate (heat-shock) method and what it is used for.
7. How can we selectively grow bacteria that have taken up the plasmid?
8. Explain the principle behind blue and white screening and its purpose in this
lab.

References
Kehoe A (1989). "The story of biosynthetic human insulin". In Sikdar SK, Bier M, Todd PW (eds.).
Frontiers in Bioprocesssing. Boca Raton, FL: CRC Press. ISBN 978-0-8493-5839-5.
https://www.sigmaaldrich.com/SE/en/technical-documents/technical-article/genomics/cloning-and
expression/blue-white-screening
https://www.nlm.nih.gov/exhibition/fromdnatobeer/exhibition-interactive/recombinant
DNA/recombinant-dna-technology-alternative.html
Khan, S., Ullah, M. W., Siddique, R., Nabi, G., Manan, S., Yousaf, M., & Hou, H. (2016). Role of 
Recombinant DNA Technology to Improve Life. International journal of genomics, 2016, 2405954. 
https://doi.org/10.1155/2016/2405954
https://www.thermofisher.com/se/en/home/life-science/cloning/cloning-learning-center/invitrogen
school-of-molecular-biology/molecular-cloning/cloning/traditional-cloning-basics.html