Lab 1

lab 1

Title: Eau that smell A synthetic biology lab for high school using BioBuilder

Objectives

By the conclusion of this laboratory investigation, the student will be able to:

• Explain how synthetic biology as an engineering discipline differs from genetic engineering.
• Explain the population growth curve of bacteria.
• Define and properly use synthetic biology terms: parts, device, inverter.
• Define and properly use molecular genetics terms: promoter, ribosome binding site, open reading frame, terminator, plasmid,
• Culture bacteria using proper microbiology methods.
• Measure the growth of a bacterial population.

Introduction

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

It has been suggested that a device that generates the banana smell during the bacteria’s log (or exponential) phase of population growth will be helpful. There are two ways to accomplish this. Both methods will continue to use the banana smell device but alter the function of the promoter. One method involves coupling the banana smell device to a new part, a log phase promoter. The other method involves using the same promoter but adding an inverter. Synthetic biologists have constructed these devices for us and transformed bacteria with them.

We have been sent three different E. coli colonies. Each contains a different device :

1. The original eau d’ Coli device
2. The banana smell generator coupled to the log phase promoter
3. The original eau d’ Coli device but with an inverter added.

Our task will be to grow these bacterial populations and test for the banana smell as the population moves through the log phase and into the stationary phase. We will determine the population growth by using a Spec 20 to measure the density of the bacteria in liquid culture. As the population increases we can assess the increasing banana smell.

Data Table

Strain	Plasmid	Colony Number on LB (if used)	Colony Number on LB + Amp	Transformation Efficiency (colonies/microgram DNA)	Color/shape/size on LB (if used)	Color/shape/size on LB + Amp
4-1	no DNA
	pPRL
	pGRN
4-2	no DNA
	pPRL
	pGRN

Sample data

Strain	Plasmid	Colony Number on LB (if used)	Colony Number on LB + Amp	Transformation Efficiency (colonies/microgram DNA)	Color/shape/size on LB (if used)	Color/shape/size on LB + Amp
4-1	no DNA	lawn	none	NA	white/cream-colored	NA
	pPRL	lawn	500	~10^3	white/cream-colored	Purple with satellites/round with crinkled edges/medium sized colonies
	pGRN	lawn	500	~10^3	white/cream-colored	Medium green with satellites, darker in the center/round with crinkled edges/medium sided
4-2	no DNA	lawn	none	NA	white/cream-colored	NA
	pPRL	lawn	none	0	white/cream-colored	NA
	pGRN	lawn	~100	10^2-10^3	white/cream-colored	Dark green with satellites/round/small

In your lab notebook, you will need to construct a data table as shown below for each of the samples. (These may be provided)

SAMPLE________________________________

Time	OD600	cells/ml*	Banana smell (+/-)
Initial
60 minutes
80 minutes
100 minutes
120 minutes
140 minutes
160 minutes
180 minutes

• 1 OD600 unit ~ 1 x 10⁹ bacterial cells/ml

Teacher Strain Table

Strain #	Plasmid	plasmid description	Cells	cells description
1-1 (NB376)	BBa_ J45250	sigma 54 directing transcription of ATF1, AmpR	BBa_J45999 (NB370)	indole- chassis, CamR
1-2 (NB377)	BBa_ J45990	sigma 54 plus tetR-4 part inverter directing transcription of ATF1, AmpR	BBa_J45999 (NB370)	indole- chassis, CamR
1-3 (NB378)	BBa_ J45200	sigma 70 directing transcription of ATF1, AmpR	BBa_J45999 (NB370)	indole- chassis, CamR
1-4 (NB379)	pUC18	no promoter, no ATF1 gene, AmpR	BBa_J45999 (NB370)	indole- chassis, CamR

Procedure

Part 1: Culturing Bacteria

We will be receiving our bacteria with the plasmid already inserted. This culture will come in the form of a "stab" or "slant", a test tube with a small amount of bacteria on a slanted media. To continue the experiment we will have to further culture the bacteria.

Day 1:

1. 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.
2. Place this culture in a 37°C incubator overnight.

This video illustrates the technique used for this transfer:bacterial plating

Day 2:

1. Using a sterile inoculating loop, transfer a bacterial colony from the petri dish to a sterile culture tube containing 5 ml of Luria Broth and 5 μl of ampicillin.
2. Place the culture tube in the roller wheel in the incubator at 37°C overnight.

Part 2: Measuring bacterial population growth

1. Prepare a sterile culture tube with

   *10 mL Luria broth
   *10 μL Ampicillin
   *10 μL isopentyl alcohol

1. Remove 5 mL of this mixture into a second sterile culture tube. Set this aside, it will be the blank for the spectroscopy.
2. Add 250 μL of bacteria from the overnight culture to the original tube. This will be the sample tube.
3. Note the time.
4. Place the tubes into the roller wheel in the incubator.
5. Prepare the spec 20 by setting it to OD600.
6. At 20 minute intervals, remove the culture tubes and transfer 750 μL of each into separate spec 20 cuvettes.
7. Read the blank and adjust the % Absorbance to zero.
8. Read the sample tube and record the % Absorbance.
9. Using proper technique, sniff the culture tube for any evidence of a banana smell.
10. Calculate the bacterial population: 1OD600 unit = 1 x 10⁹ bacteria.

Lab Report

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

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? Why are we using optical density to measure the population?
• Presume that a reader of your lab report has not read the assignment.
• Explain each phase of the bacterial population growth curve.

Methods:

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

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.

Discussion:

• Describe the results: Were we able to measure the population growth? Were we able to smell bananas?
• 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 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 genetic system, what might you construct? What would you need to do?

Citations and references:

• Be sure these are of good quality.
• Embed citations.
• Follow proper reference format.

Navigation

1. Synthetic Biology and the High School Curriculum
2. Lab 1
3. Lab 1 teacher notes
4. Lab 2
5. Lab 2 teacher notes
6. lab report rubric
7. Synthetic bio essay
8. Essay rubric
9. Synthetic Biology in High School Resources
10. National Science Engineering Standards

parts device