BioBuilder

Eau That Smell: Lab

Compare 2 competing designs to optimize system performance

Eau That Smell Lab

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 population growth curve of bacteria.
  • Culture bacteria using proper microbiology methods.
  • Measure the growth of a bacterial population:
  • Bacterial Growth Curves
  • Define and properly use synthetic biology terms:
  • Part Device Inverter iGEM
  • Define and properly use molecular genetics terms:
  • Promoter Ribosome Binding Site Open Reading Frame Terminator Plasmid

    Plasmid a circular, double-stranded DNA molecule typically containing a few thousand base pairs that replicates within a cell independently of the chromosomal DNA. Plasmid DNA is easily purified from cells, manipulated using common lab techniques and incorporated into cells

Introduction

Eau That Smell Activity

View Activity

For the 2006 iGEM competition, MIT students designed Eau d’coli, E. coli that smell like bananas when their population is in the stationary phase. They did this by inserting a device that contains a stationary phase sensitive promoter coupled to a banana smell device. The banana smell device contained a ribosome binding site (RBS), an open reading frame (ORF) that encodes the ATF1 enzyme and terminator sequences. The ATF1 enzyme converts isoamyl alcohol to isoamyl acetate, the molecule that gives bananas their characteristic smell.

In this investigation, you’ll try to generate the banana smell during the bacteria’s log phase of population growth. There are two ways (at least!) you could accomplish this. Both approaches use the original banana smell generator device (an RBS, the ATF1 gene and a transcriptional terminator). One approach couples the banana smell generator device to a new part, a log phase promoter. The other approach uses the stationary phase promoter that was used in the original MIT project but adds a genetic inverter between the stationary phase promoter and the banana smell generator device.

Eau That Smell Growth Curve

These variants have already been made for you and transformed into bacteria. In fact, you have been sent four strains of E. coli to test. Each contains a different device:

  • Sample 1-1. The original Eau d’coli device
  • Sample 1-2. The original Eau d’coli device but with an inverter added between the promoter and the RBS.
  • Sample 1-3. The banana smell generator coupled to the log phase promoter
  • Sample 1-4. A strain of E. coli that has no smell generating devices.

Your task will be to grow liquid cultures of these bacteria and measure the intensity of the banana smell as the population moves from lag phase through log phase and into stationary phase. The intensity of the banana smell can be compared to dilutions of banana extract. The population growth can be measured at each of these phases using a spectrophotometer or the McFarland Turbidity Standards.

Procedure

There are two versions of this lab. Protocol A is the shorter version, with only a measurement or two at each phase of growth. Protocol B is a longer version of this experiment, with measurements made throughout the entire population growth curve.

When you’ve finished your experiments, be sure to upload your data to the BioBuilder site to see how your measurements compare to what other BioBuilders around the country have seen. Email BioBuilder for the passcode to enter the data sharing portal.

Lab Report

Your report on this investigation may include:

  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?
    • Explain how the banana smell generator functions.
    • Why are we using optical density to measure the population?
    • Explain each phase of the bacterial population growth curve.
    • Presume that a reader of your lab report has not read the assignment.
  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.
    • Draw population growth curves of the class mean data for each sample. Indicate on each curve when you could smell bananas.
  4. Discussion
    • Describe the results: Were we able to measure the population growth? Were we able to smell bananas? Did each device produce the same results? Did the genetic systems affect the growth curve of the bacteria? Explain your answers.
    • 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 confident are you in the results? Are you equally confident in both the growth data and the smell data? Explain.
    • Is using smell to measure the banana smell valid? Why or why not?
    • What methods did you use to try to increase your confidence in the results?
    • How might we try to change this system so that we can quantify the banana smell? Would we be better off using a different kind of signal? If so, what would you suggest?
    • If you could construct a different genetic system, what might you construct? What would you need to do?
  5. Citations and references
    • Be sure these are of good quality.
    • Embed citations.
    • Follow proper reference format.

Data Sharing

When you’ve finished your experiments, be sure to upload your data to the BioBuilder site to see how your ideas compare to what other BioBuilders around the country have thought. Email BioBuilder for the passcode to enter the data sharing portal.

Acknowledgments: This lab was developed with materials and guidance from the MIT 2006 iGEM team, as well as technical insights and help from Ginkgo Bioworks