BioBuilding: Synthetic Biology for Students: Lab 1: Difference between revisions
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==Procedure== | ==Procedure== | ||
=== | ===Day 1=== | ||
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. | 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 by streaking out the stabs onto LB+amp plates. The plates will be incubated 37° overnight. <br> | ||
# 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. | # 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. | ||
# Repeat with the remaining stab samples, streaking out each onto a different petri dish. | # Repeat with the remaining stab samples, streaking out each onto a different petri dish. | ||
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This video illustrates the technique used [http://www.youtube.com/watch?v=QydH5ZoD_Aw for this transfer:] | This video illustrates the technique used [http://www.youtube.com/watch?v=QydH5ZoD_Aw for this transfer:] | ||
===Day 2:=== | |||
#Using a sterile inoculating loop, transfer a bacterial colony from one of the petri dishes to a large sterile culture tube containing 5 ml of Luria Broth and 5 μl of ampicillin. | #Using a sterile inoculating loop, transfer a bacterial colony from one of the petri dishes to a large sterile culture tube containing 5 ml of Luria Broth and 5 μl of ampicillin. | ||
#Repeat for each strain you will inoculate. | #Repeat for each strain you will inoculate. | ||
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This video illustrates the general technique for setting up [http://www.youtube.com/watch?v=0odxJy0nR9s&NR=1 overnight liquid cultures], though you’ll be transferring cells from the petri dish to the Luria Broth. | This video illustrates the general technique for setting up [http://www.youtube.com/watch?v=0odxJy0nR9s&NR=1 overnight liquid cultures], though you’ll be transferring cells from the petri dish to the Luria Broth. | ||
===Day 3: === | |||
=== | ====Procedure if using a spectrophotometer==== | ||
#Prepare a solution with | #Prepare a solution with | ||
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#Between time points, you can calculate the bacterial population: 1 OD600 unit = 1 x 10<sup>9</sup> bacteria. | #Between time points, you can calculate the bacterial population: 1 OD600 unit = 1 x 10<sup>9</sup> bacteria. | ||
====Procedure, if no spectrophotometer is available==== | |||
*The OD 600 can be estimated using the Turbidity Standards. This method uses suspensions of a 1% BaCl<sub>2</sub> in 1% H<sub>2</sub>SO<sub>4</sub> at various concentrations and is modeled after the McFarland Turbidity Scale. These suspensions appear visually similar to suspensions of E coli. | *The OD 600 can be estimated using the Turbidity Standards. This method uses suspensions of a 1% BaCl<sub>2</sub> in 1% H<sub>2</sub>SO<sub>4</sub> at various concentrations and is modeled after the McFarland Turbidity Scale. These suspensions appear visually similar to suspensions of E coli. | ||
Revision as of 09:53, 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’ 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 four different E. coli colonies. Each contains a different device: Sample 1. The original Eau d’ Coli device
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 available
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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