BioBuilding: Synthetic Biology for Students: Lab 2

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Lab 2: iTune device

  • Evaluate promoter and RBS combinations to optimize beta-galactosidase output

Acknowledgments: This lab was developed in conjunction with the MIT SEED program and with the outstanding contributions of Justin Buck, and Austin Che as well as with technical contributions from Ginkgo Bioworks



  • Explain the functioning of the lac operon and relate it to this lab.
  • Culture bacteria using proper microbiology methods.
  • Measure a kinetic chemical reaction.
  • Define and properly use synthetic biology terms: [Part,][Device,] [Measurement.]
  • Define and properly use molecular genetics terms: [Promoter,][ribosome binding site ("RBS"),] [open reading frame ("ORF"),] [Terminator,] [Plasmid.]







Part 1: Culturing Bacteria

We will be receiving our bacteria with the plasmid already inserted. This culture 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."

Day 1:

  3. Place these petri dishes media side up in a 37°C incubator overnight.

A video of this procedure is here.
Day 2:

  1. Using a sterile inoculating loop or toothpick or pipet tip, transfer a bacterial colony from the petri dish to a large sterile culture tube containing 2.5 ml of LB+Amp supplemented with 25 μL IPTG. This volume is more than enough for each strain that you or your team must grow.
  2. Repeat for each strain you will inoculate.

A video of this procedure is here.

Part 2: Beta-galactosidase assay

Procedure using a Spec 20


  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.
  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 a Spec 20 is not available

Estimate the OD 600
  3. 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.
  4. Place the card with the lines behind the standards.
  5. Make 3.0 ml of a 1:10 dilution of each cell sample, using bicarbonate buffer as the diluent.
  6. 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.
  8. Use the table below to determine the comparable OD 600.
  9. 1 OD 600 unit equals approximately 1 x 109 cells.

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 Table


    • weak
    • medium
    • strong
    • weak
    • medium
    • strong


The β-gal production is reported in Miller Units

β-gal production in Miller Units =  1000 * \frac{Abs{420}}{(t * v * Abs{600})}


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!).

Summary Data Table


When you've finished your experiments, upload your data to the BioBuilder site that's here. You'll be able to compare what you've measured to what other BioBuilders around the country have seen.

Lab Report


I. Introduction

  • Provide a brief introduction describing the field of synthetic biology.

II. Methods

  • You do not have to rewrite the procedure.
  • Explain why you did each step of the protocol.

III. Results

  • Present the data tables in clear format.
  • Create a graph summarizing the results.

IV. Discussion

  • Discuss errors and other reasons for data variability.


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