BioBuilding: Synthetic Biology for Teachers: Lab 2

{{Template:BioBuilding: Synthetic Biology for Teachers| Content= PDF of this page = Lab 2: iTune Device=

Teacher Provides

 * Tubes to grow cells
 * Eppendorf tubes or small glass tubes for running reactions
 * Cuvettes to measure absorbances if spectrophotometer is not fitted for glass tubes
 * Pipetmen and tips (P1000, P200)
 * Pipets (10 ml and 5 ml) and bulbs
 * Timers or stopwatches
 * Sharpies
 * Nitrile or Latex gloves
 * rollerwheel at 37° for growing overnight cultures of bacteria
 * vortex
 * microfuge (optional)
 * fume hood for measuring CHCl3

10 strains (see table below)

 * Store stabs at room temp
 * Store plates and liquid cultures at room temp or 4° (= fridge) for longer times.

Room Temperature

 * 500 ml LB (= 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl per liter, plus 20g of Agar for plates). Keep sterile.
 * 500 ml Z-buffer (= 8.05 g Na2HPO4*7H2O, 2.75 g NaH2PO4*H2O, 0.375 g KCl, 0.123 g MgSO4*7H2O in 500 ml H2O
 * 50 ml 1M Na2CO3 (= 10.6 g in 100 ml)
 * 10 ml 0.1% SDS (100 ul of 10% in 10 ml H2O)

4° (fridge)

 * 30 ml [|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC ONPG] (4 mg/ml in Z-buffer)
 * 2 ml [|SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Amp] (100 mg/ml in H2O, filter sterilized)
 * 20 ml [|BRAND_KEY&N4=I6758|SIAL&N25=0&QS=ON&F=SPEC IPTG] (0.1 M in H2O, filter sterilized)

Chemical Hood

 * 1 ml CHCl3

Classroom Content

 * BioBuilder material that sets up this lesson starts [[Media:BioPrimerNo2.pdf| here]]
 * Day 1: streak strains from stabs onto plates
 * Day 2: grow strains from plates as liquid overnights
 * Day 3: b-gal assays
 * Day 4: calculations of units and comparison of class data (could also be day 3 if time allows)
 * When you are done with this lab, here is a link to survey that you can offer the students. Thank you for helping us improve this content.

Annotated Laboratory Procedure
Note that these steps can be done by the students or by you (the teacher) depending on how much time and preparation you intend to take on/delegate. The only exception is the aliquot of CHCl3 (day 3) that should be done in the fume hood by the teacher. As written here, the materials are sufficient for at least 15 groups of students.

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: This video illustrates the technique used for this transfer:
 * 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) Repeat with the remaining stab samples, streaking out each onto a different petri dish.
 * 3) Place these cultures in a 37°C incubator overnight.

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.
 * 1) Using a sterile inoculating loop, transfer a bacterial colony from the petri dish to a large sterile culture tube containing 5 ml of Luria Broth, 50 μL IPTG and 5 μl of ampicillin.
 * 2) Repeat for each strain you will inoculate.
 * 3) Place the culture tubes in the roller wheel in the incubator at 37°C overnight. Be sure to balance the tubes across from each other to minimize stress on the roller wheel.

Procedure using a Spec 20
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).


 * 1) Make 3.0 ml of a 1:10 dilution of each cell sample, using Zbuffer as the diluent
 * 2) 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.
 * 3) Add 1.0 ml of Zbuffer to 11 glass spectrophotometer tubes labeled B (blank), R (reference), and 1 though 9 (the samples).
 * 4) Add 30 &mu;l of the cells (undiluted) to each tube. Add 30 &mu;l of LB to tube B, to serve as your blank.
 * 5) Next you will lyse the cells by add 60 &mu;l of 0.1% SDS and, in the hood, add 50 &mu;l of CHCl3 to each tube. Wear gloves when you add the CHCl3 and cap the tubes when you're done.
 * 6) Still wearing your gloves, vortex the tubes for 10 seconds each. You should time this step precisely since you want the replicates to be treated as identically as possible.
 * 7) Start the reactions by adding 300 &mu;l of ONPG to each tube at 15 second intervals, including your blank.
 * 8) After 7 minutes, stop the reactions by adding 750 &mu;l of Na2CO3 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 Na2CO3 makes the reactions look more yellow. The reactions are now stable and can be set aside to read another day.
 * 9) If a microfuge is available, move the contents from the tubes to microfuge tubes and spin for 1 minute at 13,000 RPM to pellet any cell debris.
 * 10) Read the absorbance of each sample tube at 420nm (OD 420). These values reflect the amount of yellow color in each tube.
 * 11) Calculate the beta-galactosidase activity in each sample according to the formula below.

Estimate the OD 600

 * The OD 600 can be estimated using Turbidity Standards. This method uses suspensions of a 1% BaCl2 in 1% H2SO4 at various concentrations and is modeled after the McFarland Turbidity Scale. These suspensions appear visually similar to suspensions of various populations of E coli.
 * 1) 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.
 * 2) Place them 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.
 * 3) 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.
 * 4) Place the card with the lines behind the standards.[[Image:Turbidity photo.jpg|400 px]]
 * 5) 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.
 * 6) 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.
 * 7) Use the table below to determine the comparable OD 600.
 * 8) 1 OD 600 unit equals approximately 1 x 109 cells.

Estimate the OD 420
Once the reactions have been stopped with Na2CO3, allow the debris to settle for a few minutes and then compare the solution's meniscus to the color samples provided. The approximate OD 420 value that corresponds to each color is listed in the table below.
 * The OD 420 can be estimated using Benjamin Moore paint chips. Color chips will be provided by your instructor.

Data Table
In 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:
 * Tested Promoter (circle the experimental sample(s) you are measuring):
 *  weak 
 *  medium 
 *  strong 
 * Tested RBS (circle the experimental sample(s) you are measuring):
 *  weak 
 *  medium 
 *  strong 

Calculations
The β-gal production is reported in Miller Units

1 Miller Unit = $$ 1000 * \frac{Abs{420}}{(t * v * Abs{600})}$$

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 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. (Use the volume of cells added to the reactions, not the final volume of the reactions themselves)

Summary Data Table
In your lab notebook, you will need to construct a data table as shown below. Fill in as many values as possible.



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

I. 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.

II. Methods

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

III. Results

 * Present the data tables in clear format.
 * Create a graph summarizing the results.

IV. Discussion

 * Draw a conclusion: Were we able to tune this system?
 * Describe the results: How do each of the promoter/RBS pairs compare? Did changing the promoters and changing the RBS have the same effect?
 * 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?

[[Media:Lab 2 data 8-11-10.xlsx| Sample Data Set]]
  TEACHERS: Note

Lab Report Rubric
Download [[Media:BioBuilding LabReport Rubric.doc| doc]] or [[Media:BioBuilding LabReport Rubric.pdf| pdf]]

Lab Report ScoreSheet
Download [[Media:BioBuilding Lab Report ScoreSheet.doc| doc]] or [[Media:BioBuilding Lab Report ScoreSheet.pdf| pdf]]

Survey Monkey Link
To help us improve the labs, you can send the students here where they can offer anonymous feedback. Thanks!

Variations to try

 * Try testing cells grown to log phase rather than stationary phase?
 * Try growing in the absence of IPTG? It's not entirely clear what lac repressor is doing in the cells anyway but the output for the devices may be different if it's not included in the growth media.
 * Try growing the cells at different temperatures? or running reactions at different temperatures?
 * If you are using the McFarland standard, would more precise or subtle standards be useful?

Feedback
We're always looking to hear back from you if you've thought about this unit, tried it, or stumbled across it and want to know more. Please email us through BioBuilder, info AT biobuilder DOT org.

Navigation
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 * BioBuilding: Synthetic Biology for Teachers
 * BioBuilding: Synthetic Biology for Teachers: Lab 1
 * BioBuilding: Synthetic Biology for Teachers: Lab 2
 * BioBuilding: Synthetic Biology for Teachers: Lab 3
 * BioBuilding: Synthetic Biology for Teachers: Lab 4
 * BioBuilding: Synthetic Biology for Teachers: Essay
 * BioBuilding: Synthetic Biology for Teachers: Design Assignment
 * BioBuilding: Synthetic Biology for Teachers: Glossary
 * BioBuilding: Synthetic Biology for Teachers: Teacher's resource room
 * BioBuilding: Synthetic Biology for Students
 * [|back to BioBuilder]