New Beginnings

Hi Friends! I'm excited to get to work on this amazing adventure with y'all! For the next few weeks I'll be in the process of gathering information of what I've done in the past with respect to sucrose selection, writing up a deeper dive lab note on the subject, and also brainstorm some additional selection markers for counterselection, useful for iterative cloning regimes like Golden Gate and Golden Braid. I'll also introduce some custom hardware I've been working on that will definitely aid in our exploration. All my work will be documented live at tinyurl.com/ATGCFFE with more polished reports written as lab notes here. The specific notion page for this project is the Sucrose Selection in E. coli and will also contain protocols in the general Flowers For Everyone lab notebook on the site linked previously. To summarize the goal of this project briefly, we want to be able to conduct routine molecular cloning without using any antibiotics. All modern molecular cloning processes, save the auxotrophic strains that require expensive media, utilize antibiotics as a means of selection. Taking a hint from the work done for auxotrophic selection in both yeast and E. coli, I came up with an idea after observing that laboratory E. coli strains lack the ability to metabolize sucrose. I found genetic data for a pathogenic E. coli strain that carried the sucrose csc operon, and isolated the coding sequences for the two critical proteins - cscA and cscB. The first gene cscA encodes a sucrose invertase which catalyzes the hydrolysis of sucrose into its constituent glucose and fructose. The liberated monosaccharides are both readily catabolized via aerobic glycolysis and act as fuel for the bugs. Without cscA, the E. coli cannot break down sucrose. The second gene, cscB, encodes a dedicated sucrose permease. This protein acts as a sucrose importer and does not require ATP to run. While digging through the literature, I found information on how sucrose sensing in E. coli functions and is actually reliant on a leaky transporter known as treB. This gene encodes an active PTS trehalose importer which requires ATP to run, but has the interesting bug-as-a-feature where it allows small amounts of sucrose to seep into the cell. This low level sucrose concentration is detected by cscR, which in the absence of sucrose acts as a repressor to the entire csc operon. When sucrose leaks in through treB and is detected by cscR, the expression inhibition of cscA (invertase), cscB (permease), and cscK (fructokinase) is lifted and the resulting proteins are produced.

Back when I first thought of this idea, I had enough budget for only one of the csc genes to be synthesized, so I decided to just print the cscA invertase and hope the seepage due to treB would be enough to feed the E. coli in media where sucrose is the sole carbon source. I cloned in a test where cscA is constitutively expressed via the strong synthetic J23100 promoter form the Anderson Collection at iGEM and grew it on M9 Minimal Media with 40mM of Sucrose added in cold (do not autoclave phosphates and sugar together; autoclave 2x conc separate and mix once cool). The following day, flasks containing E. coli carrying the cscA expression plasmid got cloudy while the ones without the invertase expression plasmid did not grow at all. I got excited and immediately made some solid M9-Suc40 media, mixing 2x molten agar with room temp 2x M9-Suc40 liquid concentrate, running positive (Glu40) and negative (no carbon) controls in parallel. I added some chlorophenol red pH indicator prior to autoclaving and adjusted the pre-clave pH to 7.0 using 1M KOH. Once the plates cooled and set, I struck the cells with or without plasmid on media containing pH indicator and M9 Minimal Media with only sucrose 40mM, only glucose 40mM, and no carbon source at all. The following day I saw faint colonies on the Suc40 plates and Glu40 pos control plates while zero growth on Suc40 plates with E. coli sans plasmid nor on the no carbon M9 plates. The colonies were a little too small to pick so I let them grow at room temp for an additional day. The following day I observed the colonies to have a yellow halo against the otherwise magenta background of the media, caused by the catabolism of glucose. The glucose only colonies had similar halo as the ones on sucrose only, but the only cells that caused the halo on the sucrose plates were the ones carrying the cscA expression plasmid. This was a nice clean experiment with good visual evidence to support my hunch that this is working.

My budget ran out back then and had to shelve the idea, but now with the amazing support from you fine folks here on Experiment, and the chronically online science twitter crew with their endless enthusiasm for my work, we can finally see this project to its conclusion, enabling students, amateurs, and researchers all over the world conduct molecular cloning experiments without the need for lifesaving and regulated frontline antibiotics. I hope to develop a protocol library, recipe cookbook, and even some strain development to compliment and support the plasmids being constructed in this project and that they all land in the hands of educators as soon as possible. Looking forward to the next months of science and discovery!