BE FPOP(Su24):BE FPOP Summer 2024
Welcome to DBE and the 20.109 lab! Get ready to dive head first into the exciting world of biological engineering as you perform your very own CRISPR genetic engineering experiment!
In this lab, you’ll get to experiment, learn, and discover alongside your peers, guided by your counselors and 20.109 lab instructors. First time in a lab? No problem! We’ll be here to guide you through all the steps and develop the skills you need to be successful in this experiment and beyond.
This is more than just a lab—it's an opportunity to see what makes biological engineering such an exciting and impactful field. You'll explore the tools and techniques that drive innovations in healthcare, sustainability, and more.
Project description
During your BE FPOP experience you will explore some of the tools and strategies that biological engineers use to harness the power of biology to solve problems. Specifically, you will test how metabolic engineering can be used to take advantage of native pathways in bacterial cells to produce commercially valuable products.
Metabolic engineering refers to the alteration of genetic and/or regulatory circuitry within organisms. The native circuitry involves a series of enzymes that perform biochemical reactions that function to convert raw substrates into products that are required for the organism’s survival. When this native circuitry is altered using metabolic engineering techniques, the goal is to use the host organisms as machines that produce valuable materials in large quantities and at low cost.
- Increasing expression of a gene that encodes an enzyme responsible for a rate-limiting step.
- Inhibiting competing pathways that divert substrate away from the pathway of interest.
- Incorporating genes from other organisms into the host.
- Altering the structure of the enzyme such that yield is increased.
To increase production of a desired product, you can either target proteins that use the substrate to generate alternate products (see A in image on right) or you can target proteins that use the desired product to generate alternate products (see B in image on right). In A the substrate is siphoned away from the reaction that generates the desired product and in B the desired product is used as substrate in a subsequent reaction. By eliminating the proteins that catalyze the reactions that result in alternate products, you can potentially increase production of your desired product.
For your project, you will manipulate the metabolism of E. coli using the CRISPRi system. Unlike more commonly used CRISPR-based technologies, CRISPRi is used to modulate expression from the genome rather than to modify the genome. This distinction is due to the use of an enzymatically-inactive dCas9 (or ‘dead’ Cas9) protein. Because dCas9 is enzymatically inactive, it is unable to cleave the DNA upon binding to the targeted sequence in the host genome. The lack of DNA cleavage results in gene silencing through impeding RNA polymerase binding, transcription factor binding, and/or transcription elongation. This method of repression referred to as CRISPRi collision.
Laboratory schedule
Day 1
| 10:00 am - 10:30 am | Presentation #1: Introduction to metabolic engineering and CRISPRi system |
| 10:30 am - 11:00 am | Exercise #1: Identifying CRISPRi targets |
| 11:00 am - 11:15 am | Demonstration: Pipetting best practices |
| 11:15 am - 12:00 pm | Exercise #2: Preparing CRISPRi experiment |
Day 2
| 10:00 am - 10:15 am | Presentation #2: Overview of method for measuring ethanol yield |
| 10:15 am - 11:15 am | Exercise #3: Measuring ethanol yield |
| 11:15 am - 11:45 am | Presentation #3: Overview of calculations for measuring ethanol yield |
| 11:45 am - 12:30 pm | Exercise #4: Analyzing your results |
