slide1
Download
Skip this Video
Download Presentation
Welcome to L319: Genetics Laboratory

Loading in 2 Seconds...

play fullscreen
1 / 25

Welcome to L319: Genetics Laboratory - PowerPoint PPT Presentation


  • 178 Views
  • Uploaded on

Yeast: S. cerevisiae. Fruit Fly: D. melanogaster. Bacteria: E. coli. Welcome to L319: Genetics Laboratory . Instructor: Dr. Amy Berndtson Assistant Instructors: Adrienne Evans (Tuesday), Ke Xu (Thursday). Yeast: S. cerevisiae. Fruit Fly: D. melanogaster. Bacteria: E. coli.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Welcome to L319: Genetics Laboratory' - zitkalasa


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

Yeast: S. cerevisiae

Fruit Fly: D. melanogaster

Bacteria: E. coli

Welcome to L319: Genetics Laboratory

Instructor: Dr. Amy Berndtson

Assistant Instructors: Adrienne Evans (Tuesday), Ke Xu (Thursday)

slide2

Yeast: S. cerevisiae

Fruit Fly: D. melanogaster

Bacteria: E. coli

Course Specifics:

Purpose of the course: Reinforce genetic principles through genetic research

Independent of L311: Topics covered in L319 are not coordinated with L311

Parts of the Course:

Lecture: Theoretical basis for experiments

Laboratory: Experiments will overlap – need to keep track of syllabus

Experiments: Designed for you to solve genetic problems - puzzles

slide4

Flowsheets: Read the Introduction handout for specific details (pp 4-5)

  • Cover Introduction and Material and Methods for a typical lab report
  • 2. Help you understand the “what and why” of an experiment
  • 3. Introductions are only due once for each experiment
  • Procedure and Rational are due each week of the lab except final data collections (see syllabus for detail)
  • 5. Flow sheets are due at the beginning of each class
  • Make copies of your flowsheets to take notes on during class
  • Concise and effective writing is rewarded!
  • Plagiarism is not tolerated
slide5

Introduction

Procedure/

Rationale

slide6

Lab Reports: Read the Introduction handout for specific details (pp 5-7)

  • Cover the Results, Discussion and Appendix of a typical lab report
  • Detailed instructions for data analysis and report components will be posted on our class web site for each lab exercise
  • Reports take effort – you may need to ask us questions
  • Don’t procrastinate until the last minute!
  • Concise and effective writing is rewarded!
  • Plagiarism is not tolerated
slide7

Exams: Read the Introduction handout for specific details (pp 7-8)

  • Based on lab report analysis – problem solving
  • 2. Will post copies of old exams
  • Lab Participation: Read the Introduction handout for specific details (p 8)
  • Based on class participation, effort and attitude
slide8

To do well in this course:

Attendance: Attend lecture and lab (require)

Flowsheets:Follow the weekly guidelines and turn flowsheets in on time

Reports: Start them early and seek help if you have questions

Exams: Make sure you understand the data analysis in each report

Lab Performance: Have a good attitude and participate

slide9

To do poorly in this class:

Attendance: Don’t attend lecture or lab (2 non-excused absence = F for course)

Flowsheets:Don’t follow the guidelines or fail to turn flowsheets in on time

Reports: Procrastinate so that you don’t have time to ask questions

Exams:Don’t understand the data analysis in each lab

Lab Performance: Have a bad attitude and don’t participate

slide10

Experiment 1A: Mutant Search in Yeast

  • I. Genetics: Variation
  • A. Wild-type alleles
  • B. Mutant alleles
  • Source of Genetic Mutation
  • A. Point mutations
  • B. Frameshift mutations
  • C. Deletions
  • D. Insertions
  • Spontaneously: occur naturally at a relatively low frequency
  • A. Error in DNA replication or repair
  • B. Deamination of the 5’-methylcytosine
  • Induced: cause mutation to occur at a higher frequency
  • A. Chemical carcinogens
  • B. UV
  • C. X-rays
  • In this experiment we will use a chemical mutagen to induce mutation in S. cerevisiae
slide11

Ethylmethane Sulfonate: alkylating agent

What kind of mutation is this?

Point mutation – transition G:A

slide12

Position of mutation within a codon:

Neutral

Acidic

Basic

How could a missense mutation affect a protein?

Could alter protein folding – protein function

slide13

Position of mutation within a codon:

How could a nonsense mutation affect a protein?

Truncates the protein – usually becomes inactive

slide14

Experiment 1A: Use EMS to induce point mutations

    • Silent (will not detect phenotypically)
    • Missense
    • Nonsense mutations
  • Experiment 2A: Use a strain of E. coli that has a high rate of spontaneous mutations
  • 1. Point mutations
  • a. Silent (will not detect phenotypically)
  • b. Missense
  • c. Nonsense
  • 2. Framshift
      • Major effect on protein function if occurs near the 5’ end of the coding sequence
      • Less effect on protein function if occurs near the 3’ end of a coding sequence or within an intron
  • 3. Deletion
  • a. Major effect on protein function if it occurs within the 5’ end of the coding sequence
  • b. Less of an effect on protein function if it occurs within the 3’ end of the coding sequence
  • 4. Insertion
  • a. Usually inactivates a protein when it inserts into both exons and introns
  • Non-revertible mutations
slide15

Life Cycle of Yeast: exist as both haploids and diploids

Mating types

We will start here

Why is it easier to find mutants?

Haploid: no other copy of an allele to mask a recessive trait

slide16

Facts about S. cerevisiae:

  • Studied for over 50 years
  • Eukaryotic Model System
  • a. Nucleus
  • b. Organelles
  • c. Exists as both haploids and diploids
  • d. Mitosis and Meiosis
  • e. 1N = 16 chromosomes
  • f. 1st genome sequenced (12.5 mega bases)
  • g. Many genes are mapped
  • 3. Isolation of mutants helped map many of the genes
slide17

Genetic Research Needs Mutants

1. Interested in a biological process

Histidine biosynthesis in yeast

2. Need mutants that have phenotypes related to the process

Histidine auxotroph

3. Need to isolate a large number of mutants because the biological process is most likely controlled by more than one gene

7 genes are involved with histidine biosynthesis in yeast

slide19

Genetic Research Needs Mutants

1. Interested in a biological process

Histidine biosynthesis in yeast

2. Need mutants that have phenotypes related to the process

Histidine auxotroph

3. Need to isolate a large number of mutants because the biological process is most likely controlled by more than one gene

7 genes are involved with histidine biosynthesis in yeast

4. Need an observable phenotype to isolate the mutants

Histidine auxotrophs will not grow in media without histidine

5. Methods of isolating the mutant cells from wild-type cells

Isolate histidine auotrophs from histidine prototrophs

slide20

What is a histidine auxotroph?

Organism (yeast cell) that cannot synthesize histidine – to survive histidine must be provided exogenously

What is a histidine prototroph?

Organism (yeast cell) that can synthesize histidine – does not need exogenous histidine to survive

slide21

What amino acid were they unable to synthesize?

How did they obtain this amino acid?

In the book Jurassic Park, the dinosaurs were auxotrophs by design

Lysine

Through plants

slide22

Can’t grow

Can’t grow

Identifying Histidine Auxotrophs with a Genetic Screen

Isolate his- mutants

slide23

Strategy to isolate histidine auxotrophs from the S. cerevisiae stain TD28

Spontaneous

Frequency of mutation (10-7)

Frequency of mutation (10-5 100 fold increase)

slide24

Spontaneous

EMS - Induced

Frequency of mutation (10-7)

Frequency of mutation (10-5))

When isolating mutants through a genetic screen you want: 100 cell/plate

How many plates would be required to isolate 1 mutant from the Unmutagenized culture?

105 plates

How many plates would be required to isolate 1 mutant from the Mutagenized culture?

103 plates

This is way too many plates to work with – impossible – too expensive

slide25

Key Steps

TD28 is auxotrophic

for Urea and Inositol

Kill 99%

Cells

Kill 90%

Cells

Why?

Decrease the number of histidine prototroph

(His+) cells

ad