Resources, Rubrics, and Nat'l/State Standards
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- 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
- Nitrile or Latex gloves
- rollerwheel at 37° for growing overnight cultures of bacteria
- 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.
- 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)
- 30 ml [ONPG] (4 mg/ml in Z-buffer)
- 2 ml [Amp] (100 mg/ml in H2O, filter sterilized)
- 20 ml [IPTG] (0.1 M in H2O, filter sterilized)
- BioBuilder material that sets up this lesson starts here
- Day 1: streak strains from stabs onto plates
- Day 2: grow strains from plates as liquid overnights
- Day 3: b-gal assays
- 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.
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.
Streak out stabs onto LB+amp plates. Incubate 37° overnight. If your class will test the whole set, there will be 10 strains to streak out.
- Dilute Amp 1:1000 in LB using sterile technique. You will need 2.5 ml for each strain you want to test, e.g. 25 ml if you will be testing all 10 strains.
- Dilute IPTG 1:100 in LB using sterile technique. You will need 25 ml for each strain you want to test, e.g. 250 ml if you will be testing all 10 strains.
- Colonies can be inoculated into the media with a toothpick, a loop, or a pipet tip.
- Grow 2.5 ml overnight cultures in large sterile glass tubes with loosely fitted caps on 37° roller wheel.
- Cultures are stable and active for a week at least (stored at room temp or in the fridge)
- Aliquot reagents for students as desired.
- It is recommended that all students or teams of students test the "reference strain" in addition to some number of experimental variants.
- Teacher should add 90 ul of the CHCl3 to 1 ml of Z-buffer for each b-gal assay to be performed. This cannot be done in bulk and then aliquoted since the CHCl3 will settle to the bottom of the tube immediately.
- At the start of class, students will dilute cells to measure their density (undiluted cell cultures are invariably too dense for spectrophotometers to read accurately), add 30 ul of undiluted cells to each assay tube that the teacher has prepared, add 60 ul SDS to the tubes, vortex, then start the reactions.
- If your teaching block is ~ 1 hour long, then the teacher can stop all the reactions by adding Na2CO3 to each tube (timing does not have to be precise to the second) and the tubes can stay at room temperature, tightly capped, overnight, or longer if you do not meet your students every day.
- This day is for reading the Abs 420 values and for doing the calculations. Ideally some class data should be pooled for some statistical analysis of accuracy and precision.
- These steps can be continued on Day 3 if you have more than 1 hour of time with the students.
- If you can, spin the contents of the reactions in a microfuge before reading the values. This will reduce cell debris and make the readings more accurate.
If a spec 20 is not available
It is still possible to conduct this lab if spec 20s are not available. The students will note that as the population of bacteria grow, the cultures get increasingly turbid.
- 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.
- 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.
The size of the tubes and the volume of sample and standard used is flexible. The important things are that the volume can obscure the thick black lines and that the samples and standards are prepared in the same fashion. See the photo below.
- 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.
- 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.
- Place the card with the lines behind the standards.
- 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.
- 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.
- Use the table below to determine the comparable OD 600.
- 1 OD 600 unit equals approximately 1 x 109 cells.
- The OD 420 can be estimated using Benjamin Moore paint chips. It is impossible to accurately reproduce these chips online. Color chips will be provided by your instructor.
- Note that the RBS makes a difference only when the promoter is weak. This can be a starting point to talk about rate determining steps and how transcription and translation are coupled in bacterial cells.
- Note that the current set of strains (2-1 through 2-9) have "weak" and "strong" promoters behaving nearly identically. There is a new set of "very strong" promoters being built that will offer a greater range of units. This can be a way to illustrate the "design-build-test" paradigm in engineering and a way to illustrate the utility of scientific understanding to help guide the redesign cycles.
Lab Report Rubric
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Lab Report ScoreSheet
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Survey Monkey Link
To help us improve the labs, you can send the students here where they can offer anonymous feedback.
Variations to try
- Measure output in log phase rather than stationary. Perhaps there will be different growth rates vs production rates?
- Model RBS strength using RBS calculator. Explore ways to model/measure promoter function. Consider other aspects of transcription/translation that may affect output.
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.
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