BioBuilding: Synthetic Biology for Students: Lab 1: Difference between revisions
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We have been sent four different E. coli colonies. Each contains a different device: | We have been sent four different E. coli colonies. Each contains a different device:<br> | ||
Sample 1-1. The original Eau d’Coli device<br> | '''Sample 1-1.''' The original Eau d’Coli device<br> | ||
Sample 1-2. The banana smell generator coupled to the log phase promoter<br> | '''Sample 1-2.''' The banana smell generator coupled to the log phase promoter<br> | ||
Sample 1-3. The original Eau d’ Coli device but with an inverter added between the promoter and the RBS.<br> | '''Sample 1-3.''' The original Eau d’ Coli device but with an inverter added between the promoter and the RBS.<br> | ||
Sample 1-4. A strain of E. coli that has no smell generating devices.<br> | '''Sample 1-4.''' A strain of E. coli that has no smell generating devices.<br> | ||
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 spectrophotometer or the McFarland Turbidity Standards to measure the density of the bacteria in liquid culture. As the population increases we can assess the increasing banana smell, comparing the smell to dilutions of banana extract. | 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 spectrophotometer or the McFarland Turbidity Standards to measure the density of the bacteria in liquid culture. As the population increases we can assess the increasing banana smell, comparing the smell to dilutions of banana extract. | ||
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#At 20 minute intervals repeat steps 11-14.<br> | #At 20 minute intervals repeat steps 11-14.<br> | ||
#Between time points, you can calculate the bacterial population: 1 OD600 unit = 1 x 10<sup>9</sup> bacteria.<br> | #Between time points, you can calculate the bacterial population: 1 OD600 unit = 1 x 10<sup>9</sup> bacteria.<br> | ||
====Procedure, if no spectrophotometer is available==== | ====Procedure, if no spectrophotometer is available==== | ||
Turbidity comparisons for some bacterial cultures (left) and McFarland standards (right)]]The turbidity of the bacterial populations can be estimated using the [http://www.microbiol.org/white.papers/WP.OD.htm McFarland Turbidity Scale]. This method uses suspensions of a 1% BaCl<sub>2</sub> in 1% H<sub>2</sub>SO<sub>4</sub> that are visually similar to suspensions of various populations of ''E. coli.''<br> | Turbidity comparisons for some bacterial cultures (left) and McFarland standards (right)]]The turbidity of the bacterial populations can be estimated using the [http://www.microbiol.org/white.papers/WP.OD.htm McFarland Turbidity Scale]. This method uses suspensions of a 1% BaCl<sub>2</sub> in 1% H<sub>2</sub>SO<sub>4</sub> that are visually similar to suspensions of various populations of ''E. coli.''<br> | ||
#Following your teacher's instructions, obtain small clear test tubes containing the turbidity standards. The tubes should contain enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom. Make sure each tube is properly labeled with its turbidity standard number. If you are filling the tubes from stock bottles of the standards, use small tubes and place enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom.<br> | |||
#Place the samples in a test tube rack that allows you to view them from the side. Use small tubes and place enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom.<br> | |||
#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.<br> | |||
#Place the card with the lines behind the standards.<br> | |||
#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.<br> | |||
#Place the sample tube next to the standard tubes. You should move the sample to compare it to the standard tubes with the most similar turbidity. You can make this assessment more precise by looking for a standard that most similarly obscures the black lines on the background card.<br> | |||
#Use the table below to determine the comparable OD 600.<br> | |||
#1 OD 600 unit equals approximately 1 x 10<sup>9</sup> cells. | |||
[[Image:McFarland_table.PNG]] | [[Image:McFarland_table.PNG]] | ||
==Data Table== | ==Data Table== | ||
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[[Image:Banana_data_table.JPG]] | [[Image:Banana_data_table.JPG]] | ||
==Lab Report== | ==Lab Report== | ||
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*What did methods did you use to try to increase your confidence in the results? | *What did 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? | *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 | *If you could construct a different genetic system, what might you construct? What would you need to do? | ||
===V. Citations and references=== | ===V. Citations and references=== | ||
*Be sure these are of good quality. | *Be sure these are of good quality. |
Revision as of 10:38, 25 August 2010
Eau That Smell Lab |
LAB 1: Eau that smell
Acknowledgements: This lab was developed with materials and guidance from the MIT 2006 iGEM team, as well as technical insights and help from Ginkgo BioworksObjectivesBy the conclusion of this laboratory investigation, the student will be able to:
IntroductionFor the 2006 iGEMcompetition, 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 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.
ProcedureDay 1We 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 by streaking out the stabs onto LB+amp plates. The plates will be incubated 37° overnight.
This video illustrates the technique used for this transfer. Day 2:
This video illustrates the general technique for setting up overnight liquid cultures, though you’ll be transferring cells from the petri dish to the Luria Broth. Day 3:Procedure if using a spectrophotometer
Procedure, if no spectrophotometer is availableTurbidity comparisons for some bacterial cultures (left) and McFarland standards (right)]]The turbidity of the bacterial populations can be estimated using the McFarland Turbidity Scale. This method uses suspensions of a 1% BaCl2 in 1% H2SO4 that are visually similar to suspensions of various populations of E. coli.
Data TableIn your lab notebook, you will need to construct a data table as shown below for each of the samples.
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
V. Citations and references
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