Laboratory 2: How do you begin to clone a gene? LSSI Alum, Lindsey Engle LSSI Alum, Shawn Hurley LSSI Alum, Mary Haus

Pre-Assignment http://www.dnaftb.org/34/animation.html Great background animated storyboard Could assign as HW if one-to-one Could also complete as pre-lab in computer lab or if you have class sets of tablets or chromebooks Could complete together in class using teacher computer https://www.youtube.com/watch?v=nfC689ElUVk **Please be aware, sticky ends are not identified correctly, but overall process is.

Resource Links/Additional Background https://www.dnalc.org/view/15476-mechanism-of-recombination-3d-animation-with-with-basic-narration.html http://highered.mheducation.com/sites/0072556781/student_view0/chapter14/animation_quiz_2.html

Important Vocabulary/Terms -Plasmid -Vector -Insert -Construct -Digest -Ligase -Palindromic -Restriction Enzyme -Sticky Ends -Complementary Bases -Origin of replication -Promoter -Antibiotic resistance gene -Regulatory gene

Complete Genetic Engineering Sequence Lab 1: Tools of the Trade – Pipetting and Gel Electrophoresis Lab 2: Restriction Digest (cut pKAN-R and pARA) Lab 3: Ligation (combine pieces to make pARA-R) Lab 4: Confirmation (gel electrophoresis) Lab 5: Transformation (introduce pARA-R into bacterial host cells) Lab 6: Culture transformed bacteria, isolate and purify the protein

Goals of Lab 2 Logistical (students will coordinate procedural steps necessary to): Perform restriction digest to generate DNA fragments for future cloning and expression of the red fluorescent protein gene in bacteria Educational (students will be able to): Identify the common characteristics of plasmids Explain how plasmids are used as vectors in gene cloning/expression Describe the function of restriction enzymes Explain restriction enzymes are used to create recombinant plasmids

What is a plasmid? Comparatively small circular double-stranded DNA molecules of bacterial origin Range in size from 1,000 to 200,000 base pairs (bp) Independent of bacterial chromosome, carrying “nonessential” genes

Antibiotic resistance Multiple cloning site (polylinker) Function: - Region responsible for initiating the copying of plasmid DNA - Site to which RNA polymerase binds to begin transcription - Gene coding for a product that confers antibiotic resistance - Unique restriction sites allow for the digestion of plasmid & introduction of insert (foreign gene) - Gene coding for a product that regulates transcription of the insert Plasmid Features: Origin of replication Promoter Antibiotic resistance Multiple cloning site (polylinker) Regulatory element(s)

promoter regulatory gene MCS (with insert) antibiotic resistance gene origin of replication (Ori)

Plasmids as Vectors http://www.pbslearningmedia.org/resource/biot11.sci.life.gen.genengdna/genetic-engineering-and-working-with-dna/

What are restriction enzymes? Catalytic proteins that function like molecular scissors, cutting double-stranded DNA at distinct recognition sites that are usually unique to a particular enzyme. http://highered.mheducation.com/sites/dl/free/0072421975/196644/restriction_endonucleases.html

What are restriction enzymes? Characteristics: Recognition sites are palindromic sequences, usually 4-8 nucleotides in length 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ Cleave covalent bonds of sugar-phosphate backbone If enzyme is a staggered cutter, generates sticky ends (unpaired overhangs capable of hydrogen bonding with complementary bases) 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ Nonemclature based on source bacterial species & strain E co R I 1st letter of genus (Escherichia) roman numeral designates order of discovery 1st two letters of species (coli) strain

Application of These Molecular Tools Scientists can build designer plasmids that contain specific restriction sites This allows scientist to cut out and recombine genes to allow for cloning and gene expression (requires cutting each DNA sample with same restriction enzyme(s)) https://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio38.swf::Early%20Genetic%20Engineering%20Experiment

Lab 2 – Creating the Digest (cutting up the DNA) Purpose: to produce the DNA fragments that will be joined to make the recombinant plasmid. Will need to cut two plasmids pKAN-R – has the rfp gene with promoter sequence (pBAD) and an antibiotic resistance gene for kanamyacin (kanR) pARA – has an antibiotic resistance gene for ampicillin (ampR) and a regulatory gene (araC), coding for arabinose activator protein Arabinose is a sugar that induces transcription of the rfp gene by working with arabinose activator protein Will use 2 restriction enzymes on each plasmid allowing the segment from pKAN-R to later be inserted into the pARA plasmid BamHI HindIII

Lab 2 – Creating the Digest

Restriction Digest Fragments Lab 2 – Creating the Digest Restriction Digest Fragments BamH I Hind III 4,706 bp BamH I Hind III 4,496 bp Hind III BamH I 806 bp Hind III BamH I 376 bp

Safety-Lab 2 Use laboratory coats, safety glasses and gloves as appropriate Avoid restrictive clothing and open-toed shoes No eating or drinking in the lab Make sure that students are familiar with the operating instructions and safety precautions before they use any of the lab equipment Check all MSDS (Material Safety Data Sheets) for all chemicals and reagents in the lab before preparing and running the lab

Lab & Aliquoting Guide-Lab 2 Reagents/Supplies Aliquot Storage Temp Notes 1.4 ml 2.5X Buffer/class (2.5xB) 20ul/group 4o   110 ul of pKAN/class (pKAN) 10ul/group -20o 110 ul of pARA/ class (pARA) 65 ul of Restriction Enzyme/class (RE) 5ul/group 12mL of DI water/ kit (dH20) 1ml/group RT Keep this for all Labs Equipment/Supplies 10 Student boxes with the following: 1 p20 micropipette 1 microfuge rack 1 p200 micropipette 1 bag of microfuge tubes 1 p1000 micropipette 1 bag of microfuge tubes 1 waste and 1 box of refillable tips (2 ul-200 ul) 1 ice bucket 4 Mini centrifuges 1 Water bath 2 Floating racks

Completing Lab 2 Teacher Tips – *make sure students label with initials/group *Remind them of mixing techniques *Make sure centrifuge is balanced before running *Identify for students when to use a new tip

Completing Lab 2 (pg. B-15 in teacher manual) Label tubes Add 4.0 uL 2.5xB into each Add 4.0 uL K into K+ and K- tubes Add 4.0 uL A into A+ and A- tubes Add 2.0 uL RE into K+ and A+ tubes and mix Add 2.0 uL dH2O in K- and A- tubes and mix Put all tubes in floating rack and set in 37oC water bath for 60 mins.* Centrifuge all tubes * - if time is limited, reducing the digest time to 20-30 minutes is aaceptable Remove and place tubes into freezer overnight

Teacher Video Resources Mixing two solutions video: https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/mixing-two-solutions Digestion video (different digest, but good techniques): https://www.youtube.com/watch?v=GsWo8dCivWs How restriction enzymes work (good, short): https://www.youtube.com/watch?v=lWXryzgRces Longer, overall of lab 2 created for absent students (screen cast): https://www.youtube.com/watch?v=4wbStjWEM8A Fun one from MIT. Covers whole process 1st half good for Labs 2&3: https://www.youtube.com/watch?v=nfC689ElUVk ***remember, sticky end issue here