BioBuilding: Synthetic Biology for Students: Lab 2: Difference between revisions
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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. Note that the volumes here are given for spectrophotometers that use glass test tubes (13x100 mm). | 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. Note that the volumes here are given for spectrophotometers that use glass test tubes (13x100 mm). | ||
# Make 3.0 ml of a 1:10 dilution of each cell sample, | # Make 3.0 ml of a 1:10 dilution (300 μL of cells in 2700 ml of Zbuffer) of each cell sample. | ||
# 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 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. | ||
# Add 1.0 ml of Zbuffer to 11 | # 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. | ||
#You can now dispose of these tubes as instructed by your teacher. | |||
# Add 1.0 ml of Zbuffer to 11 test tubes labeled B (blank), R (reference), and 1 though 9 (the samples). These are the reaction tubes. | |||
# Add 30 μl of the cells (undiluted) to each tube. Add 30 μl of LB to tube B, to serve as your blank. | # Add 30 μl of the cells (undiluted) to each tube. Add 30 μl of LB to tube B, to serve as your blank. | ||
# Next you will lyse the cells by add 60 μl of 0.1% SDS and, in the hood, add 50 μl of CHCl3 to each tube. Wear gloves when you add the CHCl<sub>3</sub> and cap the tubes when you're done. | # Next you will lyse the cells by add 60 μl of 0.1% SDS and, in the hood, add 50 μl of CHCl3 to each tube. Wear gloves when you add the CHCl<sub>3</sub> and cap the tubes when you're done. | ||
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# Start the reactions by adding 300 μl of ONPG to each tube at 15 second intervals, including your blank. | # Start the reactions by adding 300 μl of ONPG to each tube at 15 second intervals, including your blank. | ||
# After 7 minutes, stop the reactions by adding 750 μl of Na<sub>2</sub>CO<sub>3</sub> to each tube at 15 second intervals. Seven 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 Na<sub>2</sub>CO<sub>3</sub> makes the reactions look more yellow. The reactions are now stable and can be set aside to read another day. | # After 7 minutes, stop the reactions by adding 750 μl of Na<sub>2</sub>CO<sub>3</sub> to each tube at 15 second intervals. Seven 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 Na<sub>2</sub>CO<sub>3</sub> makes the reactions look more yellow. The reactions are now stable and can be set aside to read another day. | ||
# 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. It is important that you carefully pipet this mixture during this transfer such that you do not transfer any of the chloroform in the bottom of the reaction tube. The chloroform will appear as a clear layer at the bottom of the tube. | |||
# Read the absorbance of each sample tube at 420nm (OD 420). These values reflect the amount of yellow color in each tube. | # Read the absorbance of each sample tube at 420nm (OD 420). These values reflect the amount of yellow color in each tube. | ||
# Calculate the beta-galactosidase activity in each sample according to the formula below. | # Calculate the beta-galactosidase activity in each sample according to the formula below. |
Revision as of 11:47, 9 August 2011
Eau That Smell Lab |
Lab 2: iTune device
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 BioworksObjectivesBy the conclusion of this laboratory investigation, the student will be able to:
IntroductionAs 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 devices using techniques such as DNA synthesis, gel electrophoresis, polymerase chain reaction, and cloning. The synthetic biologists then test the function of the devices they’ve built, characterizing the cells that bear the devices through enzyme activity assays, fluorescent protein measurements or phenotype analysis. Depending on the device that’s being characterized, measurements may evaluate the speed of a device’s response, its sensitivity to environmental signals, or the level of a protein made by the device. It’s tempting to think that a strong quick level of response is always desired when designing genetic devices. However, depending on the role that the device will play in a system, it may be desirable to be able to tune the output to intermediate levels, or even to slow and low outputs in some cases.Tuning genetic devices may be accomplished in many ways. One method controls the rate of transcription initiation by choosing a promoter of a particular efficiency or that’s active only under some conditions, for example. Another method involves translation control, modifying the strength of the ribosome binding site to increase or decrease the translation initiation rate. Finer tuning can be achieved by rational combination of the promoter and RBS elements. Predictable design, however, is confounded by the fact that some devices are not fully insulated from others in the cell and so might be affected by the system in which they must perform. Other devices demand a lot of the cell’s resources to run and so might slow a cell’s growth or protein production rates. These problems would be like a car in which the volume button on the radio also turned the steering wheel, or like a car in which the louder you played the radio, the slower the car could run. Problematic to say the least! Thus measuring the performance of a device, even a rationally designed one, is still needed. As a starting point, we will consider a "reference device" that includes a strong log phase promoter, a strong RBS, a lacZ ORF that produces beta-galactosidase, and a transcriptional terminator sequence. Variants of this device are also available. All contain the same lacZ ORF and terminator sequence, but the devices vary in the efficiency (“strength”) of the promoters and RBSs. We will measure the output of each device, presuming that any difference in beta-galactosidase activity level will be due to the combination of promoter and RBS. The lacZ ORF provides us with an easy method to measure the activity level of each promoter/ORF combination since the beta-galactosidase that is produced by the lacZ ORF allows the bacteria to metabolize lactose (see lac operon). Normally lactose is cleaved into two monosaccharides, galactose and glucose. However, we will provide the cells with ONPG (o-nitrophenyl-β-D-galactoside) rather than lactose. ONPG will be metabolized by the beta-galactosidase into galactose and o-nitrophenol, a yellow compound. The intensity of the yellow color formed will be proportional to the amount of beta-galactosidase enzyme that the device produced in the cell. We measure intensity of yellow color using a spectrophotometer, like a Spec 20, or with visual comparisons to turbidity standards. ProcedurePart 1: Culturing BacteriaWe 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:
A video of this procedure is here.
A video of this procedure is here. Part 2: Beta-galactosidase assayProcedure using a Spec 20With 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. Note that the volumes here are given for spectrophotometers that use glass test tubes (13x100 mm).
Procedure if a Spec 20 is not availableEstimate the OD 600
Estimate the OD 420
Data TableIn 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:
CalculationsThe β-gal production is reported in Miller Units
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 TableIn your lab notebook, you will need to construct a data table as shown below. Fill in as many values as possible. Lab ReportAs you write, be sure to define and properly use all highlighted terms throughout the introduction and other parts of the lab. I. Introduction
II. Methods
III. Results
IV. Discussion
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