Lab 2

lab 2: Tuning a system

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.
• Explain the functioning of the lac Operon and relate it to this lab.
• Culture bacteria using proper microbiology methods.
• Measure the growth of a bacterial population.
• Explain the engineering paradigm and the role of tuning a system.

Introduction

As engineers, synthetic biologists engage in the “design--> build--> test” process. They design genetic devices by coupling together promoters, ribosome binding sites (RBS), open reading frames (ORF), and terminator sequences. They then build these using techniques such as DNA synthesis, gel electrophoresis, polymerase chain reaction, and cloning. The synthetic biologists then test the function of the device using methods such as enzyme assays, fluorescent… and??? During the testing phase of this process, the synthetic biologists will characterize the device. They will measure and record the speed of response and level of protein production. One may be tempted to think that when designing genetic devices that a strong quick level of response is always desired. However, given the function of the device, it may be desirable to be able to tune the output to different levels. This may be accomplished through two means. One method is using promoters that differ in efficiency which will affect the rate of transcription. The other method involves using RBSs that differ in efficiency which will affect the rate of translation. Finer tuning can be achieved by the combination of promoter and RBS chosen.

One device that has been constructed includes a stationary phase promoter, RBS, a lacZ ORF that produces beta-galactosidase , and terminator sequences. These devices all contain the same b-gal ORF and terminator sequence, so any difference in activity level will be due to the combination of promoter and RBS. This lacZ ORF provides us with an easy method to measure the activity level induced by each promoter/ORF combination. The beta-galactosidase that is produced by the lacZ enzyme allows the bacteria to metabolize lactose (see lac operon). However, if we provide ONPG (o-nitrophenyl-β-D-galactoside), it will be metabolized by the beta-gal into galactose and o-nitrophenol, a yellow compound. The intensity of the yellow color will be proportional to the amount of beta-gal that has been produced.

In this lab we measure the changes in yellow color using a spec 20 as an assay of beta-gal production controlled by each combination of promoter and RBS.

Summary Data Table

In your lab notebook, you will need to construct a data table as shown below.

Strain	Promoter	RBS	β-gal activity Miller Units (class data, may have >1 entry here)	class mean β-gal activity Miller Units
2-R	Reference promoter	Reference RBS
2-1	weak	weak
2-2	weak	medium
2-3	weak	strong
2-4	medium	weak
2-5	medium	medium
2-6	medium	strong
2-7	strong	weak
2-8	strong	medium
2-9	strong	strong

Benjamin Moore color	Paint Chip	OD 420
0330 Palm Coast Pale		0.1
0331 Lemon Souffle		0.15
0332 Banan-appeal		0.2
0333 Pineapple Grove		0.5
0334 Limon		0.7
0335 Delightful Yellow		1.0
0336 Bold Yellow		2.0

Teacher Strain Table

Strain #		Promoter	Promoter #	Artificial Units		RBS	RBS #	efficiency
2-R (= NB424)		Reference promoter	J23115	387		Reference RBS	B0035	?
2-1 (= NB425)		weak	J23113	21		weak	B0031	0.07
2-2 (= NB426)		weak	J23113	21		medium	B0032	0.3
2-3 (= NB427)		weak	J23113	21		strong	B0034	1
2-4 (= NB428)		medium	J23106	1185		weak	B0031	0.07
2-5 (= NB429)		medium	J23106	1185		medium	B0032	0.3
2-6 (= NB430)		medium	J23106	1185		strong	B0034	1
2-7 (= NB431)		strong	J23119	consensus		weak	B0031	0.07
2-8 (= NB432)		strong	J23119	consensus		medium	B0032	0.3
2-9 (= NB433)		strong	J23119	consensus		strong	B0034	1
NOTE: these promoter/RBS combinations have not been sequence verified but perform ~ as expected

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: Beta-galactosidase assay

With this assay you will determine the amount of beta-galactosidase activity associated with each sample of cells. You will perform assays of each sample in triplicate to gain some confidence in the values you measure. A table is included here to help you organize your assay, but you can make one of your own if you prefer.

1. Make 1 ml of a 1:10 dilution of each cell sample, using Zbuffer-BME as the diluent, then use 100 ul of this dilution to make another 1:10, for a final concentration of 1:100.
2. Add 400 μl of Zbuffer-BME to 13 eppendorf tubes labeled 0-12.
3. Add 100 μl of the appropriate cell dilution to each tube (1:100 of no IPTG in tubes 1-3 etc). See chart below for guidance. Add 100 ul of Zbuffer-BME to tube 0, to serve as your blank.
4. Next you will lyse the cells by add 20 μl of 0.1% SDS to each eppendorf.
5. To better lyse the cells, vortex the tubes for 10 seconds each. You should time these precisely since you want the replicates to be treated as identically as possible.
6. Start the reactions by adding 100 μl of ONPG to each tube at 10 second intervals, including your blank.
7. Stop the reactions by adding 250 μl of Na₂CO₃ to each tube at 10 second intervals once sufficient yellow color has developed. “Sufficient” is defined as yellow enough to give a reliable reading in the spectrophotometer, best between 0.3 and 1.0. Be sure to note the time you are stopping the reactions. Also be sure to remember that adding the Na₂CO₃ makes the reactions more yellow.
8. When all your samples have been stopped, add 250 μl of Na₂CO₃ to the blank and spin all the tubes in the microfuge for 1 minute at 13,000 RPM to pellet any cell debris.
9. Move 0.5 ml of each reaction to plastic cuvettes and read the absorbance at 420nm. These values reflect the amount of yellow color in each tube.
10. Read the absorbance of each at 550 nm. These values reflect the amount of cell debris and differences in the plastic cuvettes themselves.
11. Calculate the beta-galactosidase activity in each sample according to the formula below:
    • Beta-gal Units = Abs at 420 minus (1.75 times Abs at 550nm) all divided by the product of time in minutes, volume of cells (from original culture) in ml and OD600 (of the original culture), then all times 1000.
12. Dispose of your samples properly.

Data Table

In your lab notebook, you will need to construct a data table as shown below.

• Tested Promoter (circle one):
  
  • weak
    
  • medium
    
  • strong
    
• Tested RBS (circle one):
  
  • weak
    
  • medium
    
  • strong
    

Sample number	Strain	Abs 600	Start time	Stop time	Time elapsed (minutes)	Abs 420	β-gal activity Miller Units
B = blank	none		0:00
R	Reference strain		0:15
1	2-1		0:30
2	2-2		0:45
3	2-3		1:00
4	2-4		1:15
5	2-5		1:30
6	2-6		1:45
7	2-7		2:00
8	2-8		2:15
9	2-9		2:30

Calculations

The β-gal production is reported in Miller Units

1 Miller Unit = [math]\displaystyle{ 1000 * \frac{(Abs{420} - (1.75*Abs{550}))}{(t * v * Abs{600})} }[/math]

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.

Abs 550 is the Spec 20 absorbance at 550 nm. It is a measure of cell debris.

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

t is the reaction time in minutes.

v is the culture volume in mls.

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? 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.

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.

• Create a graph summarizing the results.

Discussion:

• Draw a conclusion: Were we able to tune this system?

• Describe the results: How do each of the promoter/RBS pairs compare?

• 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?

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