The Jellyfish Gene. Cloning Genes & Transformations. Heredity & Human Affairs (BIO-1605) Spring 2012

Transcription

1 The Jellyfish Gene Cloning Genes & Transformations Heredity & Human Affairs (BIO-1605) Spring 2012

2 Recall DNA Structure DNA is comprised of nucleotides (sugar, phosphate, base) linked together & arranged into a double helix. 2

3 Recall Chromosome Structure DNA is very long Compacts into chromosomes to fit into cells Topoisomerases wind & unwind DNA 3

4 Recall Gene Structure Gene: basic unit of heredity Sequence of bases (A, T, G, C) that encodes a specific protein Introns & exon exons encode the protein products; introns are removed by splicing after transciption 4

5 DNA in Bacteria Chromosomal DNA compact double helix Plasmid DNA relaxed double helix 5

6 Genes in Plasmids Genetic engineering: Inserting (or changing) a new gene into a DNA molecule is called cloning. A plasmid can be used to clone a gene (e.g. insulin, GFP). Cloning Vector 6

7 Cloning of a Gene: general step-by-step 1. DNA molecules are cut with restriction enzymes that recognize a specific sequence. 2. The gene of interest is inserted into plasmid DNA ( sticky ends ). 3. DNA is put back together using DNA ligase. 4. Recombinant DNA is inserted into bacteria host cell. 7

8 How Do You Put the Recombinant DNA into Bacteria Cells? TRANSFORMATION 8

9 Genes into Plasmids: Transformation g5cdv6xoivx4xewg AbBv1KWiIjs=&h=350&w=400&sz=94&hl=en&start=2&itbs=1&tbnid=nvSoOpZrkRlSM:&tbnh=109&tbnw=124&prev=/images%3Fq%3Dplasmids%26hl%3Den%26gbv%3D2%26ndsp%3D20%26tbs%3Disch:1 Cloned genes integrated into plasmids are taken in by bacteria cell through transformation. Plasmid DNA is copied by the host cell; each daughter cell receives copies of the plasmid when cell division occurs. New cells, with new genes, can now produce new products (proteins). 9

10 Transformation One day before transformation experiment: grow single isolated colonies of bacteria. This is the host cell. We are using E. coli (Jaime started these yesterday) Don t use smears of bacteria! These are NOT isolated colonies! Isolated colonies = circles of bacteria 10

11 Transformation Important Steps 1. Make (E. coli) cells competent (using CaCl 2 & ice/heat shock) 2. Plasmid DNA (pgreen) must remain intact until added to the test tube containing competent cells. 3. pgreen DNA must pass across E. coli cell membrane. 4. pgreen DNA must be expressed by the host cells. 5. Transformants, cells expressing the new DNA, are selected for using LB agar + AMP (ampicillin antibiotic) & can be viewed 11 using ultraviolet light.

12 Why did we choose the pgreen plasmid for our transformation? AMP gene: Ampicillin resistance gene It s easy to test for successful transformants due to the presence of these 2 genes (AMP, GFP). GFP gene: Green Fluorescent Protein 12

13 REASON 1: Why did we choose the pgreen plasmid? GFP gene comes from an aquatic organism, Aequorea victoria, a bioluminescent jellyfish. It glows in the dark! When the GFP gene is isolated from the jellyfish, inserted into the pgreen plasmid, then incorporated into our E. coli cells, they will glow too! We ll test to see if our bacteria cells were transformed by: 1. Noting the color of the new colonies (should be yellow-green). 2. Checking for colonies to glow in the dark under UV light. 13

14 Fun Facts About GFP! GFP can be used as a marker to study medical ailments including: Cancer Tumor formation Virus infection and spread Confirm the ability to clone organisms Genetics like stem cell development 14

15 For example To study the adenovirus: (presents as cold symptoms) 15

16 For example To study Feline Immunodeficiency Virus (FIV): (Similar to the HIV virus) 16

17 For example To study stem cells (Pig stem cells are very similar to human) 17

18 For example To study reproduction (Completed at UPenn) 18

19 For example To cut loose after a long day in lab Genetically altered GFP to glow colors other than green! :D 19

20 REASON 2: Why did we choose the pgreen plasmid? pgreen plasmid also contains a gene for AMP resistance. When the AMP gene is inserted into the host cell s DNA, these E. coli cells become resistant to the antibiotic ampicillin. We ll also test to see if our bacteria cells were transformed by: 1. Noting which plates (+ amp or amp) have bacterial growth after the transformations. 2. Bacteria that can grow on +amp plates now have the amp gene from the pgreen plasmid (i.e. were transformed). 20

21 Today s Procedure: Part 1: Start with the Edvotek Plasmid DNA Kits. This model will help you learn more about plasmids & cloning. Do How Do I Clone a Gene simulation exercises 1 6 & answer related questions. 21

22 Today s Procedure: Part 2: Transformations - Instructor will lead you through the procedure. Be sure to read & follow directions precisely as written on your student directions sheet. Before doing one step, make sure to read the next step so that you know what to do next! Keep all items sterile. Work in groups of 2. 22

23 Today s Procedure: *Special Notes* (Very important - follow all directions to the letter ) *Step 5: Use isolated colonies only. *Step 5: transfer bacteria, not agar don t scrape too hard! *Step 6: Immediately & completely suspend the cells in the CaCl 2. *Step 11: Label plates (on bottom, not lid) according to the instructions. *Step 13: Heat shock the cells. Take your ice bucket to the 42 0 C water bath. Do not carry tubes in room temperature. The temp change must be abrupt! 23