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iTune Device

Evaluate promoter and RBS combinations to optimize beta-galactosidase output

OBJECTIVES

By the conclusion of this laboratory investigation, you should be able to:

  • Explain how synthetic biology as an engineering discipline differs from genetic engineering.
  • Explain the engineering paradigm and the role of tuning a system.
  • Explain the functioning of the lac operon and relate it to this lab.
  • Culture bacteria using proper microbiology methods.
  • Measure a kinetic chemical reaction:
  • Measurement
  • Define and properly use synthetic biology terms:
  • Part Device Inverter
  • Define and properly use molecular genetics terms:
  • Promoter Ribosome Binding Site Open Reading Frame Terminator Plasmid

INTRODUCTION

Some Biodesign Principles

As engineers, synthetic biologists engage in the design–> build–> test cycle. They designgenetic parts, devices and systems. For example a bacterial gene expression device might be designed by thoughtfully coupling together a promoter, a ribosome binding site (RBS), an open reading frame (ORF), and a terminator sequence. Synthetic biologists can also build the devices they design, using techniques such as DNA synthesis, gel electrophoresis, polymerase chain reaction, and cloning. To close the loop on the design/build/test cycle, it’s important for synthetic biologists to have good ways to test the function of the cells they’ve built. This might mean characterizing the cells behavior with enzyme activity assays, fluorescent protein measurements or phenotype analysis. Depending on what the system is suppose to do, measurements might be made to test the speed of a device’s response, its sensitivity to environmental signals, or its level of a protein output.

It’s tempting to think that a strong quick response is always going to be the “best” when designing genetic systems, devices or parts. However, depending on the particulars of the design specification, the output might need to be held at intermediate levels, or even to slow and low outputs in some cases.

There are a few places in the DNA –> RNA –> protein pathway that let us “tune” the output. It’s possible to control the rate of transcription initiation, choosing a promoter that’s active only under some conditions, for example. Translation control is another way to control the output, increasing or decreasing the translation initiation rate by modifying the sequence of the ribosome binding site for instance.

Finer tuning can be achieved by rational combination of promoter and RBS elements, but it’s not trivial to predictably design this way. Some devices end up dependent on others in the cell (they are not fully insulated) and other devices demand a lot of the cell’s resources, slowing a cell’s growth rate if they really demand a lot. Imagine a car in which the volume button on the radio also turned the steering wheel, or a car in which the louder you played the radio, the slower the car could run. This is the current state we face when we engineer genetic systems, devices and parts…problematic to say the least!

About your experiment

Understanding the performance of a device, even a rationally designed one, is needed to reliably engineer genetic systems. Your measurements will compare a designed device to a reference one made from a strong log phase promoter, a strong RBS, a lacZ ORF that produces beta-galactosidase. The designed variants in your experiment all contain the same lacZ ORF, but the devices vary in the efficiency (“strength”) of the promoters and RBSs. You will measure the output of each device, presuming that the combination of promoter and RBS can explain any differences detected in measured beta-galactosidase activity.

The lacZ ORF can be used to measure the activity level of each promoter/RBS combination since the beta-galactosidase enzyme that is encoded by the lacZ ORF allows the bacteria to metabolize lactose (look up information about the lac operon if you need a refresher about this). Normally lactose is cleaved into two monosaccharides, galactose and glucose. You will provide the cells with ONPG (o-nitrophenyl-β-D-galactoside) rather than lactose. When the ONPG reacts with the beta-galactosidase enzyme, the ONPG gets converted into galactose and o-nitrophenol, a yellow compound. Because the intensity of the yellow color is proportional to the amount of beta-galactosidase enzyme in the cells, the intensity of the yellow color measures the output of the promoter/RBS/lacZ ORF combination. You can measure the intensity of the yellow color using a spectrophotometer, like a Spec 20, or with visual comparisons to yellow paint-chip standards.

PROCEDURE

DAY 1

Your teacher will inform you if you are performing this part of the protocol or if it has been done for you.

We will be receiving our bacterial strains with the plasmids already inserted. The strains may come in the form of a “stab” or “slant,” a test tube with a small amount of bacteria on a slanted media, in which case you will have to streak out the bacteria onto a petri dish to continue the experiment.

If the bacteria have arrived on petri dishes, you can proceed to Day 2.

Procedure

Using a sterile toothpick or inoculating loop, gather a small amount of bacteria from the stab and transfer it to a petri dish containing Luria Broth (LB) agar plus ampicillin medium.

  1. Repeat with the remaining stab samples, streaking out each onto a different petri dish.
  2. Place these petri dishes media side up in a 37°C incubator overnight.

Watch how you can restreak the strains.You should wear gloves though!!

DAY 2

Your teacher will inform you if you are performing this part of the protocol or if it has been done for you.

If the liquid overnights have already been grown, you can proceed to Day 3.

Procedure

  1. Using a sterile inoculating loop or toothpick or pipet tip, transfer a bacterial colony from one of the petri dishes to a large sterile culture tube containing 3 ml of Luria Broth (LB) + ampicillin supplemented with 25 μL IPTG.
  2. Repeat for each strain you will inoculate.
  3. Place the culture tubes in the roller wheel in the incubator at 37°C overnight. Be sure to balance the tubes across from each other to minimize stress on the roller wheel. Alternatively, tubes can be placed on a platform shaker to grow the cells with aeration overnight.

Watch how you can grow liquid overnights of the strains.You should wear gloves though!!

DAY 3

With this assay you will determine the amount of beta-galactosidase activity associated with each sample of cells. As a class you should try to perform replicate assays of each sample (so each strain gets measured two or three times) and then pool your class data to gain some confidence in the values you measure. A data table is included to help you organize your assay, but you can make one of your own if you prefer.

PROCEDURE IF USING A SPECTROPHOTOMETER

Note that the volumes here are given for spectrophotometers that use glass test tubes (13×100 mm).

  1. Make 3.0 ml of a 1:10 dilution (300 μL of cells in 2.7 ml of bicarbonate buffer) of each cell sample.
  2. If you made the dilution in glass spectrophotometer tubes, you can proceed to the next step. If not, you will need to transfer some of this diluted cell mixture to a cuvette or glass spectrophotometer tube. The exact amount to transfer will depend on the size of the cuvette you use. Your teacher will provide further instructions.
  3. Measure the Absorbance at 600 nm (OD 600) of this dilution. Record the value X 10 in the data table. This is the density of the undiluted cells. If you do not have a spectrophotometer and are using Turbidity Standards instead, follow the instructions in the next section.
  4. You can now dispose of these dilutions and tubes as instructed by your teacher.
  5. Add 1.0 ml of bicarbonate buffer to 11 test tubes labeled B (blank), R (reference), and 1 though 9 (the samples). These are the reaction tubes.
  6. Add 100 μl of the cells (undiluted) to each tube. Add 100 μl of LB to tube B, to serve as your blank.
  7. Next you will lyse the cells by add 100 μl of dilute dish soap to each tube.
  8. Vortex the tubes for 10 seconds each. You should time this step precisely since you want the replicates to be treated as identically as possible.
  9. Start the reactions by adding 100 μl of ONPG to each tube at 15 second intervals, including your blank.
  10. After 10 minutes, stop the reactions by adding 1 ml of soda ash solution to each tube at 15 second intervals. Ten minutes is sufficient time to provide results that are yellow enough to give a reliable reading in the spectrophotometer, best between 0.1 and 1.0. Usually this color is approximately the same as that of a yellow tip for your pipetman. Don’t be surprised when the soda ash makes the reactions look more yellow. The reactions are now stable and can be set aside to read another day.
  11. If you conducted the reaction in glass spectrophotometer tubes (your teacher will tell you this), you can skip to the next step. If not, you will need to transfer some of the reaction mixture from the reaction tubes to a cuvette or glass spectrophotometer tube. The exact amount to transfer will depend on the size of the cuvette you use. Your teacher will provide further instructions.
  12. Read the absorbance of each sample tube at 420nm (OD 420). These values reflect the amount of yellow color in each tube. If you do not have a spectrophotometer and are comparing the color to paint chips instead, follow the instructions in the next section.
  13. Calculate the beta-galactosidase activity in each sample according to the formula below.

PROCEDURE, IF NO SPECTROPHOTOMETER IS AVAILABLE

PART 1: Estimate the OD 600

The turbidity of the bacterial populations can be estimated using the McFarland Turbidity Scale. This method uses suspensions of a 1% BaCl2 in 1% H2SO4 that are visually similar to suspensions of various populations of E. coli.

    1. Remove 2 ml from each sample to read lag phase density of each. If you are testing all 4 samples you should now have 4 small test tubes.
    2. Following your teacher’s instructions, obtain small clear test tubes containing the turbidity standards. The tubes should contain enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom. Make sure each tube is properly labeled with its turbidity standard number. If you are filling the tubes from stock bottles of the standards, use small tubes and place enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom.
    3. Place the standards in a test tube rack that allows you to view them from the side. Use small tubes and place enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom.
    4. On a blank index card or paper use a marker to draw two thick black lines. These lines should be within the height of the standards.
    5. Place the card with the lines behind the standards.
  1. Make 3.0 ml of a 1:10 dilution of each cell sample, using bicarbonate buffer as the diluent.
  2. To compare your bacterial cultures to the standards, you will need to place the bacterial sample in a test tube of the same size and equal volume as the standards. Be sure to label these sample tubes.
  3. Place the sample tube next to the standard tubes. You should move the sample to compare it to the standard tubes with the most similar turbidity. You can make this assessment more precise by looking for a standard that most similarly obscures the black lines on the background card.
  4. Use the table below to determine the comparable OD 600.
  5. 1 OD 600 unit equals approximately 1 x 109 cells.

PART 2: Estimate the OD 420

The OD 420 can be estimated using Benjamin Moore paint chips. Color chips will be provided by your instructor.

  1. Once the reactions have been stopped with soda ash solution, allow the debris to settle for a few minutes and then compare the solution’s meniscus to the color samples provided. The approximate OD 420 value that corresponds to each color is listed in the table below.
  2. Calculate the beta-galactosidase activity in each sample according to the formula below.

DATA COLLECTION AND ANALYSIS

Construct a data collection table

In your lab notebook, you will need to construct a data table as shown below. If you are testing only a subset of the promoter and RBS collection, be sure to note which ones you are investigating:

Tested Promoter (circle the experimental sample(s) you are measuring):

  • weak
  • medium
  • strong

Tested RBS (circle the experimental sample(s) you are measuring):

  • weak
  • medium
  • strong
PERFORM CALCULATIONS

The β-gal production is reported in Miller Units

β-gal production in Miller Units = notation

Where:

Abs 420 is the Spec 20 absorbance at 420 nm. It is a measure of the yellow color produced by the β-gal activity. It is a unitless number.

Abs 600 is the Spec 20 absorbance at 600 nm. It is a measure of the cell density. It is a unitless number.

t is the reaction time in minutes.

v is the volume of cells added to the reaction in mls. (Not μl!).

CONSTRUCT A DATA SUMMARY TABLE

In your lab notebook, you will need to construct a data table as shown below. Fill in as many values as possible.

LAB REPORT

As you write, be sure to define and properly use all highlighted terms throughout the introduction and other parts of the lab.

  1. Introduction
    • Provide a brief introduction describing the field of synthetic biology.
    • Briefly describe the purpose of the lab. What are we trying to do here? Presume that a reader of your lab report has not read the assignment.
    • Discuss the function of the promoter and the RBS. Relate your discussion to the function of the lac operon.
  2. Methods
    • You do not have to rewrite the procedure.
    • Explain why you did each step of the protocol.
  3. Results
    • Present the data tables in clear format.
    • Create a graph summarizing the results.
  4. Discussion
    • Draw a conclusion: Were we able to tune this system?
    • Describe the results: How do each of the promoter/RBS pairs compare? Did changing the promoters and changing the RBS have the same effect?
    • Analyze the data: Be sure to discuss how each part of the experiment adds to your conclusion.
    • Discuss errors and other reasons for data variability.
    • How might experiments like this one help us learn about evolution?

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