diff --git a/content/Biokemi/Plasmidlabb/Plasmid Lab Part 1 OCR.md b/content/Biokemi/Plasmidlabb/Plasmid Lab Part 1 OCR.md new file mode 100644 index 0000000..982ba2c --- /dev/null +++ b/content/Biokemi/Plasmidlabb/Plasmid Lab Part 1 OCR.md @@ -0,0 +1,219 @@ +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 + diff --git a/content/Biokemi/Plasmidlabb/Plasmid Lab Part 2 OCR.md b/content/Biokemi/Plasmidlabb/Plasmid Lab Part 2 OCR.md new file mode 100644 index 0000000..9c2ec44 --- /dev/null +++ b/content/Biokemi/Plasmidlabb/Plasmid Lab Part 2 OCR.md @@ -0,0 +1,187 @@ +Recombinant DNA technology +Lecture by: +Anne Wöhr +anne.wohr@gu.se + +Molecular Cloning +→ Performed beforehand (not done during this lab) + +Molecular Cloning +→ Performed beforehand (not done during this lab) +We aim to isolate/separate +these two plasmids to +select the recombinant +plasmid and to verify the +presence of the gene of +interest + +Day 1: Transformation of competent cells +→ performed during this lab (day 1) + +Day 1: Selection of transformed cells +→ performed during this lab (day 1 + day 2) + +Day 2: Picking & expansion of blue +and white colonies +→ performed during this lab (day 2) + +Revision +Blue-white screening +Plasmid: +• AmpR: Ampicillin resistance (β-lactamase) +• LacZ: α-peptide for functional β-galactosidase +enzyme +• BamHI restriction site in LacZ gene for +insertion of DNA +Growth medium: +• Ampicillin: Only successfully transformed +bacteria carrying plasmids can survive in the +presence of ampicillin +• IPTG : activates transcription of the LacZ gene +by binding its repressor +• X-gal: β-galactosidase degrades X-gal. The +product has a blue colour! + +Day 3 - work overview +✓ Purify plasmids +✓ Restriction enzyme digestion +✓ Run on agarose gel +✓ Interpret results + +Day 3: Material +Spin column +Tubes labeled with +A1; A2; A3; A4; H2O +Plasmid preparation kit +Collection tube & +spin column (blue) + +Day 3: Plasmid purification +→ performed during this lab (day 3) +Buffer A1 (Cell Suspension) +Tris/HCl (pH 8.0), EDTA, RNase A +Buffer A2 (Cell Lysis) +NaOH; SDS +Buffer A3 (Neutralization/Binding) +Contains acetate and guanidine +hydrochloride +Buffer A4 (Wash, reconstituted) +Contains ethanol, NaCl, EDTA, and +Tris/HCl + +cells grown in LB- +media overnight +Transfer 2x 750 µl into +microcentrifuge tube +Bacterial pellet = cells + plasmid +→ Discard superatant +Balance the centrifuge +Day 3: harvest cells & purify plasmids +A1 - resuspension buffer +A2 – cell lysis buffer +A3 – neutralization/ +binding buffer +Plasmid in supernatant +cell debris as pellet +transfer supernatant +to column +Plasmid binding +Discard +flow-through +Wash with A4 +Transfer column to new tube +add H2O to elute plasmid + +QIAprep Miniprep Handbook, Appendix A: Background Information, Preparation of cell lysates, p 43 +Plasmid purification +Buffer A1: +Bacterial cells are resuspended in a buffer containing Rnase A. +Buffer A2: +Bacteria are lysed under alkaline conditions (NaOH). SDS solubilizes the phospholipid +and protein components of the cell membrane +Lysis and release of cells contents. Alkaline conditions: denaturation of chromosomal +and plasmid DNA as well as proteins. +Buffer A3: +The lysate is neutralized and adjusted to high-salt-binding conditions. The high salt +concentration causes denatured proteins, chromosomal DNA, cellular debris, and SDS +to precipitate, while the smaller plasmid DNA renatures correctly and stays in solution. +DNA is bound to silica membrane of spin columns in high-salt buffer. RNA, cellular +proteins and metabolites are not retained on the membrane. +Buffer A4: +Washing and reconstitution of DNA. Salts are efficiently removed by this wash step. +H2O: +The purified plasmid DNA is eluted from silica membrane by addition of water. The +elution is pe is dependent on a low salt concentration and a stable pH (pH 7-8.5). + +Restriction enzyme working solution: +Restriction enzyme buffer +H2O +Restriction enzyme (keep it cold!) +Add restriction enzyme to a portion of eluted plasmid +KEEP THE REST OF UNDIGESTED PLASMID AS CONTROLS FOR +LATER USE Incubate at 37°C for 60 min +Day 3: Restriction enzyme digestion + +Day 3: Restriction enzyme digestion +Plasmid without insert (2700 bp) +Plasmid with insert in BamHI site (4200 bp) +Cut with BamHI → bands at 2700 bp and 1500 bp + +• Samples are mixed with 6x loading +dye to make them ”heavier” to stay +in wells +• Separation of DNA molecules +based on their size +• DNA negatively charged +• Agarose gel for separation +• Shorter molecules move faster and +migrate farther than longer ones +• Visualization of DNA with SYBR +safe DNA stain +Day 3: Agarose Gel Electrophoresis + +GeneRuler 1kb DNA ladder = size marker +marker +2700 bp +1500 bp +4200 bp +Day 3: Expected Results + +• Relaxed/linear: intact circle but “nick” in one strand +• Linear: both strands are cut (at the same location) +• Supercoiled: fully intact with both strands uncut, appears in a compact +form +Plasmid conformation affects migration + +Lab schedule +✓ Purify plasmids +✓ Restriction enzyme digestion +1-2h incubation time → lunchbreak and +everyone will be back at the same time +✓ Run on agarose gel +approximately 1h → go through the +expected results to be able to ask +appropriate questions +✓ Interpret results +make sure to ask a lot of questions while you have +the chance!!! + +Lab reports +✓ Write according to the guidelines on the handout on Canvas +✓ One lab report per group (Names and group number on the cover page) +✓ In English +✓ Upload your lab reports on CANVAS, deadline 07/12/2025 + +Lecture Questions +1. What are the sites the plasmid contains that allow for this experiment? +2. What are the 3 compounds in the LB plates that allow for selection of +bacteria with plasmid and the distinction of plasmids with and without +the inserted gene? +3. What are the six steps in plasmid preparation and purification? +4. What is a restriction enzyme and why was it used in this experiment? +5. Explain the principle of Gel Electrophoresis, what is it for? +6. What´s the compound that allows for the visualization of the DNA in +the gel? +7. How many times does BamHI cut the plasmid without the insert? And +the plasmid with the insert? +8. Explain the different plasmid conformations that exist. \ No newline at end of file