July 1st - 2024
*I'll be copypasting my lab notes from my Notion notebook here for convenience. Please comment below with questions! Do follow along live updates at www.tinyurl.com/ATGCFFE and all data files at www.tinyurl.com/ATGCSWEET. Enjoyyyy.*
Today marks the start of the project as of the Experiment.com fundraise. I’m spending the day organizing my work and thoughts to pick up where the project left off prior to the grant and generally trying to plan out the next few months. The first and foremost deadline is to have a draft plasmid working by August, and by that I mean good progress toward completely decoupling from antibiotics in the cloning process. The deadlines are arbitrary and soft but I’m hoping to keep to them as best as I can, with what little spare time I have for this project.
So far I have made the above plasmid which overexpresses the cscA sucrose invertase gene from a wild E. coli, which is a synthetic coding sequence synthesized via Twist Bioscience and codon optimized for E. coli K-12 expression via the VectorBuilder Codon Optimization Tool. This plasmid acts like a Golden Braid compatible expression vector which takes any L0 coding sequence and expresses it under strong constitutive expression via the J23100 synthetic promoter from the Anderson Collection and the BioBrick B1006 hairpin terminator. Any coding sequenced flanked by BsaI cut sites with the AATG 5’ overhang and CGCT 3’ overhang will clone in seamlessly and express continuously. Handy tool!
I transformed this plasmid into NEB Turbo cells back in January and plated it on M9 Minimal Media, where Sucrose 20mM was the sole carbon source and it grew remarkably well. Inside the media I added some Chlorophenol Red to detect any acidification of the media as a result of the breakdown of glucose, which would only happen if the sucrose was hydrolyzed by the cscA invertase. It worked!
Just to be sure I was not fooling myself, I went ahead and bought all the E. coli strains Carolina Biological Supply had in stock to test if any native sucrose metabolism was present. I compared growth in M9 Minimal Media with Glucose as well as with Sucrose and let them shake in a 24 well plate with 2mL of media, overnight at 37C.
Nothing grew on the sucrose plates despite a heavy spike of cells in each well. I then decided to repeat the experiment, including the NEB Turbo cells transformed with the ECXK-cscA plasmid under same conditions and media then followed by an endpoint addition of Universal pH Indicator Solution to see if any metabolism was afoot.
The metabolism was varied in glucose but the most important result was the cscA well in sucrose which showed a VERY STRONG pH drop indicating active sucrose metabolism. The spike of cells was heavy and we do see some clouding in the sucrose wells of W3104, C, and OP50 cell lines though this could have been from the initial spike. Regardless of the inconsistency in growth, the cscA cells in glucose AND sucrose grew nicely. This was the convincing point for me where I decided to pursue the experiment in full.
Fast forward to the present day and the above plasmid is what I am aiming to create. I call it pDonutMk1, after a Twitter Poll for naming the construct was conducted, and it consists of an origin of replication (truncated and modified) from pUC19, and eforCP Red Fluorescent Protein expression circuit using the J23100 promoter and B1006 terminator as a visual marker, and a hacked together selection marker where I replaced a typical kanamycin resistance (nptII) coding sequence with my cscA sucrose invertase. I really don’t know how strong the kanR promoter is or if it will be enough, but let’s see if this works. It also does not have a formal terminator, as most of the kanR workhorse plasmids floating around do not have one (more on that later) but I left room for two unique cut sites to add a proper terminator later.
To construct this prototype plasmid, I’m going to steal the eforCP circuit from the Rainbow Chromoprotein Plasmid Collection I sell on my web store (shameless plug) and PCR the visual marker + ori with Bsa1 cut sites on either side. Then I’ll do an enzyme digest of both that PCR amplicon and the cscA coding sequence from the ECXK-cscA plasmid in a MoClo reaction to make the final construct. The real concern will be that the plasmid will no longer have any antibiotic selection marker and so I would have to develop a transformation protocol for transforming only using M9 minimal media and NO EXTRA CARBON SOURCE. Recovering cells is gonna be interesting, lol.
This is essentially building the airplane while I’m flying it but I’ll do some preliminary experiments to get transformation working in minimal media using the original ECXK-cscA plasmid and shore up the protocol. Likely I’ll use the calcium chloride method because its cheap and easy to source (road salt here in the US) and fairly simple to prep compared to my usual TSS buffer. Maybe I can tweak the TSS buffer recipe to make a minimal media version and compare transformation efficiencies.
Last but not least I need to select a strain of E. coli that will be my workhorse going forward as the NEB Turbos are a proprietary cell line and I’d be looking into selling kits at some point for convenience. All in all I am excited to start tinkering toward getting these plasmids into the hands of educators around the world. Yay, frugal science!
0 comments