Homework
Weekly homework submissions:
Week 1: Principles and Practices
Class Assignment First, describe a biological engineering application or tool you want to develop and why. This could be inspired by an idea for your HTGAA class project and/or something for which you are already doing in your research, or something you are just curious about. I am interested in engineering bacteriophage (viruses which infect bacteria) in order to detect harmful bacterial pathogens by using the engineered viruses to deliver and express a certain reporter gene upon infection.
Week 10 - Advanced Measurement & Measurement Technology
Class Assignment Waters Part I - Molecular Weight We will analyze an eGFP standard on a Waters Xevo G3 QTof MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the native and denatured (unfolded) states. The conditions for LC-MS analysis of intact protein cause it to unfold and be detected in its denatured form (due to the solvents and pH used for analysis).
Week 11 - Bioproduction & Cloud Labs
Class Assignment
Class Assignment Part I. Example design performed in Benchling found here: Design for lab found here: Part II. Performed in lab. Resulting Gel Image here:
Class Assignment Python Script Uploaded to form and shared with lab. Post-lab questions I investigated the paper “Slowpoke: An Automated Golden Gate Cloning Workflow for Opentrons OT-2 and Flex” as I was interested in using GGA to assemble my engineered phage satellite for my final project and was curious as to how I could integrate an Opentrons workflow into the process. The paper tested two GGA kits, the MoClo Yeast Toolkit (YTK) and the SubtiToolKit (STK), with GFP as the reporter for both, then transformed the assembled plasmids into E. coli and Saccharomyces cerevisiae (yeast) or Bacilius subtilius based on the according kit. The authors found high assembly and transformation efficiencies using the opentrons to complete the protocols (17/17 colonies and 8/13 colonies contained the GGA construct with GFP using the YTK and STK kits respectively).
Class Assignment Part A. How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons) 500 grams is approximately 3.01e26 Daltons, which converts to 3.01e24 molecules of amino acids.
Class Assignment
Class Assignment DNA Assembly What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? We used NEB’s Q5 2X Hot Start Master Mix for our lab, but like the Phusion MM, it contains a DNA polymerase (for the extension step of PCR), as well as dNTPs and Mg2+ as polymerase cofactors and a buffer.
Class Assignment IANNs What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? IANNs can account for greater complexity, such as changing the level of output based on gradients or combinations of signals, which better reflects realistic biological systems.
Class Assignment General Qs Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production. Describe the main components of a cell-free expression system and explain the role of each component. Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why. How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup. Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each. Kate Adamala Qs Design an example of a useful synthetic minimal cell as follows: