Laboratory 3: building a recombinant plasmid LSSI Alum, Lindsey Engle LSSI Alum, Shawn Hurley LSSI Alum, Mary Haus

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 3 Logistical (students will coordinate procedural steps necessary to): Synthesize recombinant plasmid (pARA-R), possessing the red fluorescent protein (rfp) gene, ampicillin resistance gene (ampr) and regulatory gene (araC), for future cloning and expression Educational (students will be able to): Explain how DNA ligase is used to create a recombinant plasmid Describe possible recombinant plasmids that form when ligating a restriction digest

What are Ligases? Enzymes that function like molecular glue, bonding two separate pieces of DNA together. Ligation involves forming covalent bonds in the sugar-phosphate backbone generating a contiguous piece of DNA. Role in Gene Cloning: When the unpaired bases of two sticky ends come together and form hydrogen bonds, the DNA ligase catalyzes the formation of the covalent bonds between adjacent nucleotides.

What are Ligases?

Lab 3 – Building the Recombinant Plasmid Purpose: to ligate the DNA fragments you produced during Lab 2 to make new recombinant plasmids. There are four fragments that will be recombining There are 10 different possible plasmids produced, each with different combinations (have students predict all possible combinations – key is in teacher guide) The plasmid of interest is the one containing rfp, pBAD, ampR, and araC The recombinant plasmid is called pARA-R

Recombinant plasmid of interest

Safety-Lab 3 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 3 Reagents/Supplies Aliquot Storage Temp Notes 30 ul Ligase/class (Lig) 2ul/group -20o   50 ul 5xB (Ligase B)/ class (5xB) 5ul/group 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 3 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 3 (pg. B-33 in teacher manual) ***complete paper plasmid activity (see next slide) Check reagents Place K+ & A+ tubes into 70oC water bath for 30 min. to inactivate restriction enzymes* Label LIG tube with group and class period DIY Water Bath * - If time is limited, 90oC for 5-10 min. is acceptable

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

Completing Lab 3 (continued from previous slide) Remove K+ and A+ from 70oC water bath Add 4.0 uL K+ into LIG Add 4.0 uL A+ into LIG Add 3.0 uL 5xB into LIG Add 2.0 uL dH2O into LIG Mix reagents in LIG Place LIG, K+, and A+ in rack Centrifuge LIG tube Incubate at room temperature overnight

Teacher Resources Making a recombinant (good, short clip): https://www.dnalc.org/view/15476-Mechanism-of-Recombination-3D-animation-with-with-basic-narration.html Recombinant DNA animation: http://www.bioteach.ubc.ca/TeachingResources/Applications/GMOpkgJKloseGLampard2.swf Overall process – Good short animation: http://www.pbslearningmedia.org/resource/biot11.sci.life.gen.genengdna/genetic-engineering-and-working-with-dna/ Recombinant DNA quizlet: https://quizlet.com/7435136/recombinant-dna-technology-flash-cards/

Note on Sequencing Suggested sequences are included in your teacher guides, lessons today were not necessarily presented in this order. Lab 2 Sequence (pg. B-4) Session 1 – Review, questions, and discussion Session 2 – “Clone that Gene” paper plasmid activity and questions Session 3 – Complete lab 2 Lab 3 Sequence (pg. B-24) Session 1 – Review and questions Session 2 – Complete lab 3