Assignment EightSaccharomyces cerevisiae Growth Characterization II:

Differential Growth Curves & Spectrophotometry

Functional Genomics Research Stream • Freshman Research Initiative • by Dr. Patrick J. Killion

Assigned: March 10, 2009

Due: March 24, 2009

Submission: Beginning of Lecture, 2:30 PM

Format: This Printout

General Introduction

IMPORTANT NOTE: Background material and explanation is purposefully omitted from this assignment text. For more background on the subject matter covered in this assignment be sure to see the lecture notes and the online course notebook, each found on the course website (course materials): http://fg.cns.utexas.edu/

Time Considerations

Please be aware of the time constraints of the various sections and plan your attack accordingly.

Section A - Background Information

In this assignment you will be growing two different strains of Saccharomyces cerevisiae:

1. S288C (wild type)2. ∆CST6

Question 1: What is CST6 (what are we describing here)?

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Question 2: Per our previous lectures and discussions of yeast strains, describe the strain ∆CST6?

__________________________________________________________________________________________

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Question 3: Where might you go (on the Internet) to learn more about the function or role of CST6 (one website)?

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Question 4: From that website: What MOLECULAR FUNCTION does CST6 play?

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Question 5: From that website: What BIOLOGICAL PROCESS does CST6 play a role in?

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Question 6: Another word for gene deletion is null. Now that you know this, is there anything known about the growth rate of the growth rate of ∆CST6? What about over-expression of the gene?

null: ________________________________________________________________________________

over-expression: ______________________________________________________________________

Question 7: What is the Systematic Name and Aliases of CST6?

__________________________________________________________________________________________

Section B - Differential Growth in Liquid Media

Introduction

This portion of the assignment is going to teach you how to do two things:

1. Use the 20D+ Spectrophotometer to measure cell density in culture.2. Use the NanoDrop ND-1000 to measure cell density in culture.3. Perform a growth curve characterization of multiple strains of yeast.

Chapter 2 of your course textbook has an amazing amount of background material on spectrophotometry. You should really consider reading most of it right about now.

Are you done with that? Did you pay particular attention to concepts of transmission and absorbance?

Let us now move on to how to actually use the spectrophotometer we have in the laboratory. Chapter 9 of the online course notebook has significant information on the use of the 20D+ Spectrophotometer and NanoDrop - both of which inform you as to how to measure cell culture density which will be important in the completion of this assignment.

Using the Spectrophotometers

Equipment

The work this week introduces two new pieces of equipment: the Spectronic 20D+ and the NanoDrop. Both are equipment commonly found in modern biology laboratories.

The procedures for using each of these pieces of equipment are documented in Chapter 9 of the online course notebook. There are laptops conveniently located next to each of these pieces of equipment. Please - no gloves on the keyboards!

Spectronic 20D+ Spectronic 20D+ Cuvettes NanoDrop ND-1000

Rumor has it this piece of equipment was a critical component in the discovery of the new world.

These are the glass cuvettes that are used with our spectrophotometer. Do not attempt to use any other cuvette.

The greatest thing since Rush. Once you see the beauty of the NanoDrop you will un-friend the Spectronic.

Performing a Growth Curve

This information, as well, is covered in Chapter 9 of the online course notebook. A growth curve is the monitored and quantified growth of any cell culture over a period of time. You start some cells growing and on regular intervals you take a measurement of cell density. At the end of the growth you are able to plot this gathered data as a curve.

General Procedure

1. Start a 5 mL culture of YPD and incubate the cells overnight at 30°C.For assignment below you will be using a common stock so running an overnight culture is not needed.

2. The next day, take an OD 600 measurement.See protocol for Using the 20D+ Spectrophotometer.

3. Calculate the amount of overnight culture required to start a new 40 mL YPD culture at an OD600 of 0.10 using the equation S1×V1=S2×V2. Normally cultures are begun at OD600 0.05 but for this assignment we are going to start the culture off with a higher cell density (0.10).

4. Combine this calculated amount of overnight culture with fresh YPD to start a new culture at OD600 of 0.10.

5. Take an OD600 reading. This is “Hour 0” (actual measurement).

6. At regular intervals (varies per organism and strain) take OD600 measurements on both cultures and continue to do so until the growth rate levels off.

Typical Graphical Output

The following graph represents the typical growth of yeast or bacteria in liquid media.

Question 8: Why does the growth take off rapidly from time 2 to 6 hours?

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Question 9: What is this time-period usual referred to as (2 to 6 hours)? There is a specific term.

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Question 10: Why are cells usually captured during this time-period (2 to 6 hours) for experiments or measurements?

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Question 11: Why does the growth begin to slow after 6 hours?

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Question 12: Why does the growth reach a peak around 11 hours and begin to drop thereafter?

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Question 13: Will all growth curves look like this (changes will always occur at these hour marks)?

YES or NO and WHY: __________________________________________________________________

__________________________________________________________________________________________

__________________________________________________________________________________________

Experiment

In this section you will be growing two different strains of Saccharomyces cerevisiae:1. S288C (wild type)2. ∆CST6

You will be growing both over a period of 6 hours and capturing data with both the Spectronic 20D+ and NanoDrop ND-1000 spectrophotometers so as to render a growth curve for each strain. Please note that as you use both spectrophotometers - the expectation is that both devices will provide very similar values for a time-point. If they do not you should stop, think, read, and perhaps ask for assistance (after the stop, think, read steps).

You will be using a provided common overnight set of cultures in the incubator: S288C (common), ∆CST6 (common) and thus you will begin the general procedure (above) at step (2).

Collect the following data:

Device Strain Hour 0 (Target)

Hour 0 (Actual)

Hour 1 Hour 2 Hour 3 Hour 4 Hour 5 Hour 6

Spectronic S288C OD 600 ~0.10

NanoDrop S288C OD 600 ~0.10

Spectronic ∆CST6 OD 600 ~0.10

NanoDrop ∆CST6 OD 600 ~0.10

Graphical Output

Excel Plots

On a single graph plot the growth curve for both S288C and ∆CST6 as recorded by both spectrophotometers.

This means that one single line plot will have four curves (two strains as measured by two devices). Your plot should include a legend to clearly differentiate the four curves. The plot should have a clear title and each axis clearly labeled.

Print the graph and staple it to the end of this assignment. You do not need to submit your Excel file this week.

Question 14: Did both strains seem to grow the same?

YES or NO and WHY: ___________________________________________________________________

___________________________________________________________________________________________

___________________________________________________________________________________________

Question 15: At what time point did each strain begin to exit log-phase growth (or did they)?

S288C: _______________________________________________________________________________

∆CST6: _______________________________________________________________________________

Section C - Differential Growth on Plates

Introduction

Plate spotting is a technique used to study the growth of a yeast on a solid surface (a plate).

It is typically used to study the relative growth of multiple yeast strains on one common growth surface. Serial dilutions of each culture are performed such that each row represents a strain while each column represents a dilution. Dilutions are used because it can be difficult to separate a weakly growing strain from wild type at high cellular concentrations. This is done by taking a yeast culture (or multiple), making a series of serial dilutions, and plating them (in “spots”) on a plate. For the example below we are giving details for plating one strain (a row). The procedure would be performed multiple times for each strain that needs to be co-plated.

Procedure

First you should make or retrieve the plates on which you will be plating your cells. Next you need to prepare 100 µL serial dilutions of an overnight cell culture.

You will likely want to create the following tubes (for each strain, thus eight tubes total):

‣ Tube 1 - 1/1

‣ Tube 2 - 1/10

‣ Tube 3 - 1/100

‣ Tube 4 - 1/1000

1. Take 20 µL from Tube 1 and pipette it onto the plate.Try and place the drop about half an inch from the center and toward the left side of the plate as you are going to place the next two drops to the right of this one and in a straight line across the plate.

2. Take 20 µL from Tube 2 and pipette it onto the plate about half an inch to the right of the spot from Tube 2.

3. Do the same for Tube 3, and place it about the same distance from the Tube 2 spot as is the Tube 2 spot from the Tube 2 spot.

4. You can use this plate to measure as many dilutions as you wish so long as you space them out and keep them in sequential order.

5. Place the plate in the appropriate incubator for growth.

After your plates grow overnight they will look something like the figure below.

‣ The columns correlate to tubes (Tubes 1, 2, 3, and 4 in this case).

‣ The rows are different strains grown at the same time.

‣ One might say that strains A, C, and D grew better than strains B and E.

‣ Are you able to see it (especially in the last dilution)?

Experiment

In this section you will be plate spotting two different strains of Saccharomyces cerevisiae:1. S288C (Wild Type)2. ∆CST6

You will need two YPD plates:1. Both strains will be plate spotted (two rows) and grown at 30°C.2. Both strains will be plate spotted (two rows) and grown at 37°C (incubator PAI 2.14).

You will be using a provided common overnight set of cultures in the incubator: S288C (common), ∆CST6 (common).

From each of these cultures take 1 mL and make the following 1 mL dilutions:

S288C:

‣ Tube 1 - 1/1

‣ Tube 2 - 1/10

‣ Tube 3 - 1/100

‣ Tube 4 - 1/1000

∆CST6:

‣ Tube 1 - 1/1

‣ Tube 2 - 1/10

‣ Tube 3 - 1/100

‣ Tube 4 - 1/1000

Plate and grow the dilutions such that (top row & blue = S288C dilution series, bottom row & red = ∆CST6):

Grow one plate in the PAI 2.46 incubator (30°C) and one plate in the PAI 2.14 incubator (37°C) overnight.

Question 16: Did the strains show differential growth at 30°C? (YES or NO)

Question 17: Did the strains show differential growth at 37°C? (YES or NO)

Question 18: What were your expectations for both temperature conditions and did the results match them?_________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Sign Off On Plate Growth:

Present your plates to a research staff member and get a signature. Research Staff: ________________________

Assignment Submission

Sign Off

When you believe you have the assignment completed ask a staff member (Dr. Killion, any Functional Genomics Research Stream Undergraduate Mentor) look over your work and sign below.

You should be able to present the staff member:‣ Complete laboratory notebook with all tables called for in this assignment.

What to do with your materials:‣ Discard your liquid cultures (correctly).‣ Place your plates in the new 4°C refrigerator.

Date: ______________________________________________

Time: ______________________________________________

Staff Name: __________________________________

Staff Signature: __________________________________

Submission

This assignment is due on March 24, 2009 - 2:30 PM.

This printed document is the only component that is needed for final submission.

Please answer the following questions:

I worked a total of _____ hours on this assignment. (please be honest, not graded for hours)

I worked with a group of the following people:

_____________________________________ _______________________________________

_____________________________________ _______________________________________

I adhered to the group work policy of the research stream:

Signature: _______________________________________ Date: ________________________________