iTune Device

Evaluate promoter and RBS combinations to optimize beta-galactosidase output

This lab focuses on predictable design. Students will examine the role of parts, such as promoters and ribosome binding sites, in determining the output of a genetic device. The students measure b-galactosidase enzymatic activity as the device’s output, enabling them to look through the lens of molecular genetics to evaluate a device’s behavior. Students study transcription, translation, the lac operon and enzyme function within a synthetic biology context. Part 1 of the lab offers an opportunity for the students to develop microbiology skills, though this procedure may be done by the teacher if time is an issue. In Part 2, the students lyse the cells to release the β-galactosidase enzyme and then carry out a timed enzymatic reaction, later calculating the enzyme activity associated with each genetic construct.

SAMPLE UNIT PLANS

Some helpful background content and skills

Content

DNA Basics: what it is and how it works
Gene Expression: Transcription, Translation, Operons
Recombinant DNA: Restriction enzymes, cloning, selection with antibiotic resistance

Skills

Pipetting
Measuring cell density with a spectrophotometer
Basic microbiology skills: Culturing bacteria, Aseptic technique, Antibiotic resistance, Population dynamics

SOME LAB MATERIALS TO HAVE ON HAND

HARDWARE: Some basic biology laboratory equipment such as glassware, pipets, a water bath or heat block to heat samples, an icebucket or styrofoam cup to cool samples, 4C fridge and ice-cold freezer (ideally to -20C) to store samples, a stir plate to mix samples, and an incubator to grow samples (though room temperature can work too). Some additional experiments might need a chemical hood, electrophoresis chambers, spectrophotometer, or a PCR machine.
CONSUMABLES: Some basic consumable laboratory equipment such as pipet tips, plastic tubes, growth media, petri dishes, toothpicks, spreaders, sterile loops, bleach or the like for decontamination and disposal of bioreagents. Some additional experiments might need additional consumables such as gel running buffers, loading buffers, stains to visualize DNA or proteins, cuvettes, PCR master mix, or an autoclave for sterilizing reagents.

RESOURCES AND DOWNLOADS

Specific materials for teaching this lab

CLASSROOM RESOURCES

READINGS

PPTs

VIDEOS

HANDOUTS

LAB RESOURCES

LAB MATERIALS

VIDEOS

PPTs

OTHER RESOURCES

Workflow for iTune Device Protocol

ANALYSIS AND FEEDBACK

Lab Follow-ups

Students can compare their results to others using an alternative lysis protocol. Download protocol that uses CHCl3.

Students can rehearse or repeat the procedure to determine if timing of each step is as precise as possible.

If a microfuge is available, students can compare their OD420 results before and after spinning the stopped reactions for one minute to pellet any cell debris.