Laboratory 2A: How do you begin to clone a gene? LSSI Alum, Shawn Hurley

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 2A: Restriction Digest (cut pARA-R) Lab 4A: Confirmation (gel electrophoresis) Lab 5A: Transformation (introduce pARA-R into bacterial host cells) Lab 6: Culture transformed bacteria, isolate and purify the protein

Goals of Lab 2A Logistical (students will coordinate procedural steps necessary to): Perform restriction digest on recombinant plasmid (pARA-R) in order to indirectly confirm its identity and possession of the red fluorescent protein gene 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 how 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 2A – Digesting pARA-R (cutting the recombinant plasmid) Purpose: to produce DNA fragments of appropriate size that will confirm the presence of the rfp gene. Will need to cut recombinant plasmid (construct) pARA-R – has the rfp gene with promoter sequence (pBAD), an antibiotic resistance gene for ampicillin (ampr) and a regulatory gene (araC) that codes for arabinose activator protein - Arabinose is a sugar that induces transcription of the rfp gene by working with arabinose activator protein Will use two restriction enzymes on the construct, liberating the rfp insert from the remainder of the plasmid/vector BamHI HindIII

Recombinant plasmid (pARA-R)

Expected Resulting Digest Fragments Lab 2A – Digesting pARA-R Expected Resulting Digest Fragments BamH I Hind III 4,495 bp Hind III BamH I 807 bp

Safety-Lab 2A 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 2A Reagents/Supplies Aliquot Storage Temp Notes 1.4 ml 2.5X Buffer/class (2.5xB) 20ul/group 4o   110 ul of recombinant plasmid (RP)/ class (pARA-R) 10ul/group -20o 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 2A 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 2A (pg. C-15 in teacher manual) Label tubes (“R+” & “R-”) Add 4.0 uL 2.5xB into each Add 4.0 uL of RP into each tube Add 2.0 uL RE into R+ tube and mix Add 2.0 uL dH2O in R- tube and mix Centrifuge all tubes Put all tubes in floating rack and set in 37oC water bath for 60 mins.* Remove and place tubes into freezer overnight * - if time is limited, reducing the digest time to 20-30 minutes is aaceptable

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

Paper Plasmid Activity Modeling Activity: Choose the appropriate restriction enzyme(s) to use to insert the vasopressin gene into a plasmid-based vector Recombinant DNA Synthesis Handout (PDF) (PPT) – These may take several minutes to download