Bacterial Transformation With pGFP Genetic Engineering: Bacterial - PowerPoint PPT Presentation

Bacterial Transformation With pGFP

Genetic Engineering: Bacterial Transformation A technique used often in a biotechnology or molecular lab.

Watch this phenomenon: First 1 minute of this video: https://www.youtube.com/watch?v=VP cdF4Cx_tI

I NOTICE… On your index card, write down, I notice…

Watch this phenomenon… First 45 seconds https://www.youtub e.com/watch?v=n0 UzdYRnMtY&t=12s

I WONDER… On your index card, write down, I WONDER…

SCIENCE IS OBSERVING… SCIENCE IS WONDERING… This week, we will introduce you to a genetic engineering experiment where you can be a scientist with a fun lab.

WHAT IS GENETIC ENGINEERING? Genetic engineering is to manipulate genes in cells using biotechnology to make something practical or useful. What can you think of, that has been made or modified, that is useful or helps us?

Food and Medicines made from Genetic Engineered Products Rennet found cheese: used for coagulating and curdling cheese Humulin: a genetically engineered product that lowers glucose/sugar level in the blood.

Vaccines and Cancer Drugs produced from Genetic Engineering Vaccines: for polio, flu, HiB, etc Herceptin: a drug that combats breast cancer

Textiles made from spider silk!

“Meat” Products made from Plants

But why make something green? “Fluorescent tags” or labels are used to study proteins or gene expression.

Microtubule movements of a cell: https://www.youtube.com/watch?v= lHKdrDe5pwM

Fluorescent label organelles of a cell Image from: https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescent-proteins

WHAT ARE THE STEPS IN GENETIC ENGINEERING? The first step is bacterial transformation. By the end of this week, you can be a genetic engineer too!

Genetic Engineering with Green Fluorescent Protein + =

A Jellyfish named Aequorea victoria

GFP

Green Fluorescent Protein In Nature Why GLOW?

Green Fluorescent Protein In Nature: Why GLOW? • Defense: Startle predators • Feeding: Lure prey • Mating: Lure a mate • Camouflage • Subtle communication • others (?)

Can we make white bacteria turn GREEN??? + =

GFP Expressing Animals

Purified GFP What ?

Green Fluorescent Protein Gene DNA Sequence ATCGATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCGT • CAATTGTCTGATTCGTTACCAATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGG AACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACAT TGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCC TCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAA ACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCG CGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAA GCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGT CGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAA GCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGAC GACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCT GATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGAT AAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCC GGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTC CATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCT TATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCAC GGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATT AGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGGCTAGAAATA ATTTTGTTTAACTTTAAGAAGGAGATATACATATGGCTAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCA ATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCTA CATACGGAAAGCTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACT ACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGC CATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAA GTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACAT TCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGA ATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAA TACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAA

Definitions – A Reminder • DNA is an information storage molecule: it carries the information to make all the parts of living thing. • DNA is a code for a “parts list” for a living thing • DNA is universal – almost all living things use it for inheritance. • A Gene is a piece of DNA that carries the information to make a “part”

New Definition: Plasmids • A Plasmid is a small circular piece of DNA used to transform cells • There are 2 genes on this plasmid that are important

2 Important Genes • 1. Ampicillin Resistance Gene: Ampicillin is a deadly antibiotic to bacteria! Killing off non-transfomed cells, cells that did not get the pGFP plasmid DNA: that means most of the bacteria will be killed off. • 2. GFP Gene: so our cells will have the DNA information to make a green protein and turn green!

Green Fluorescent Protein Gene pGFP This is your armor that resists the antibiotic! Ampicillin Resistance Gene (antibiotic can’t kill it)

Bacterial Transformation http://www.dnai.org/b/index.html DNAi.org à Manipulation à techniques à Transferring and Storing à Transformation

Summary of Procedure

Summary of Procedure 1. Scoop up some bacteria with a sterile loop and mix bacteria well in ice cold Salt Water (CaCl 2 ) 2. Add 10 ul of pGFP plasmid DNA ul = micro liter = one millionth of a liter! A very small amount of liquid!) 3. Keep cells COLD !! On ice for 20 minutes

4. : place cells in warm water for exactly 50 seconds (42° centigrade is 5° warmer than Body Temperature, which is 37°) 5. Place tubes back on ice 6. Feed cells with a nutrient broth (LB) and allow them to recover for 10 minutes 7. Plate cells on the appropriates dishes.

Reasons for Each Step • Incubate on ice slows down the cell membrane • Heat-shock Increases pores or holes in cell membranes to allow plasmid DNA to enter the cells • Nutrient broth incubation Feed the cells: allows cells to recover from heat shock

Precise Measurement is Critical!

Procedure • Please See Pages 6 – 8 of your Handouts

We Gratefully Acknowledge These Resources for Images Figure 1: http://wonderfulanimals.blogspot.com/2012/06/aequorea-victoria.html • Figure 2: http://www.tamabi.ac.jp/idd/shiro/light/luminesence/organelle.html • Figure 3: Bio Rad Education • Figure 4:https://www.pinterest.com/pin/113645590567113482/ • Figure 5: http://slideplayer.com/slide/6625882/ • Figure 6: https://en.wikipedia.org/wiki/Green_fluorescent_protein • Figure7: http://www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/Stem-Cell- • Haematopoiesis/Brachyury-GFP-model Figure 8: Bio Rad Education • Figure 9: http://2009.igem.org/Team:Utah_State/Secretion • Figure 10: http://www.seasky.org/deep-sea/bioluminescence.html • Figure 11: http://www.tsienlab.ucsd.edu/Images.htm • Figure 12: http://slideplayer.com/slide/6829297/ • Figure 13: Bio Rad Education •

We Gratefully Acknowledge These Resources for Images Figure 14http://microbesrule.blogspot.com/2014/04/introducing-freshman-to- • transformation.html Figure 15 - 21: Bio Rad Education • Figure 22: BABEC.org •