Week 2 HW: DNA Design

Part 0: Set up in Kernel

Repo is titled CBillingsley. My homework folder is titled Billingsley-HTGAA_Project.

Part 1: Define your expression target

The molecules that I am expressing are 5 separate enzymes: protease, amylase, lipase, cellulase, and pectinase. The sequences will come from the UnitProt database, with the ID numbers listed in the answer to part 2. The chassis is a cell-free system, derived from an E. coli cell lysate. The end product will ultimately be an application of the direct cell-free reaction, so the product(s) do not need to be secreted or targeted anywhere other than the traditional mixture.

Part 2: Design your genetic parts

For each of my five constructs, I used: Promoter BBa_J23101. This is from the iGEM Part Registry. The reason this was selected was because it is a standard constitutive promoter that can be expressed in E. coli and does not require any sort of induction or modulation to function. RBS BBa_B0034, a standard strong E. coli RBS. This is from the iGEM Part Registry. I chose this as a standard strong RBS. This seems like it will translate to my 5 different enzymes. Terminator BBa_B0015. This is from the iGEM Part Registry. I chose this as a standard Terminator that seems to be effective for simple constructs and will translate well to a simple cell-free expression constructs.

For my 5 constructs, I did use ChatGPT to help me find 5 well-characterized bacterial enzymes that would help give me a good starting point for my project. Protease: Subtilisin, UniProt P04189 Amylase: Alpha-amylase, UniProt P00691 Lipase: Lipase A / EstA, UnitProt P37957 Cellulase: Endoglucanase EglS, UnitProt P10475 Pectinase: Pectate lyase, UnitProt P39116

Part 3: Assemble your construct

I followed the generic description for each of my 5 constructs, including going from promoter → standard RBS → [variable CDS] → standard strong terminator. Because I am not yet at the point where I am thinking about how I will put the enzymes into my cocktail and if they might have different promoters/relative strength TX/TL machinery, the only difference between the constructs is their CDS, which is a E. coli codon optimized version of the UniProt protein sequences.

Part 4: Optimize and quality-check

Codon-optimize: Because I started from the UniProt protein sequences, all of my constructs were codon optimized already. Since I will have a cell free lysate, I chose not to worry about restriction sites. Sequence search: Yes, when I blast my sequences, they return the same sequences that ChatGPT helped me to elucidate for the project. Safety Screen: It looks like the sequence safety scanner does a sequence comparison for flagged sequences of concern. Luckily, nothing flagged for me, since I am using 5 well-characterized, safe bacterial enzymes.

Part 5: From design to build

Since my constructs are relatively small and simplistic, I believe that it would be the most cost effective to order a pool of oligos from a synthetic DNA maker. If I needed more constructs, I could create promoters and amplify the constructs through PCR too. My sequence does not contain certain sequences that might be hard to synthesize due to the inevitable simplicity of a cell-free protein synthesis system of this scale. While I would potentially test my idea by synthesizing a bunch of oligos in a one pot reaction, I would have to start the separate them out and dose them in various concentrations to start to mimic the precise control that I would like to have over the reaction and the cocktail of enzymes for proper cleaning efficiency.

Part 6: What don’t you know yet?

I think one of the biggest unknowns that I have yet to figure out is the fact that once an active protease in my reaction, it may start digesting your other four enzymes or even components of the lysate. However, proteases are such important components of cleaning stains that we get on our clothes, so I will have to try to work with my concoction. To check if this happens, I would be able to run a CFPS reaction with and without the protease construct being dosed, and I will evaluate what the end titer is of the reaction and verify if other proteins have been degraded with a technique such as SDS PAGE. The second unknown is whether or not I will want to have different construct architecture for my different enzymes (e.g. each has a different promoter of a varying strength). I have already decided that I would like to vary the concentrations of these different types of enzymes in my end product, and I know that previous literature has identified that you can vary construct concentration to achieve this end. However, I will want to run various CFPS reactions with different constructs having different promoters and evaluating if I see a resulting difference in protein concentration.