Start Date July 1st. 2024 - Sebastian S. Cocioba - Binomica Labs
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This journal, along with all my other work, lives at tinyurl.com/ATGCFFE
I ordered primers for the citrate idea, L0 cloning and detection primers, from Elim Biopharma and should arrive by Thursday.
I also ordered parts to build my MiniSpec Mk6 8-channel turbidometer system which I will discuss in a later lab journal entry. This device can measure up to 8 batch cultures of 15mL and measure their optical density every 1 second continuously. This will be extremely useful in measuring the fitness of microbes engineered with various selection markers to determine the best expression elements empirically.
After mulling over some ideas I think I came up with a solid counterselection system to compliment sucrose positive selection via cscA — CITRATE! This idea stems from the need to have two different selection markers to be able to use the iterative Golden Braid System I am so fond of.
Addgene: Orzaez Lab GoldenBraid 2.0 Kit
In short, the system uses four plasmids and each pair (alpha and omega) can recursively clone together into one of the plasmids of the opposite pair. Two alpha plasmids’ transcription units can be chained together when cloned into an omega plasmid. The resulting omega plasmid carrying the two alpha transcription units can then be cloned along with a second omega plasmid into a chain in an alpha plasmid. This cycle can iterate forever for as long as your plasmid can replicate and DNA prep process can maintain plasmid size without shearing.
Alpha 1 + Alpha 2 → Omega 1 Omega 1 + Omega 2 → Alpha 1
This system relies on the ability to counter select between the alpha and omega plasmid pairs. Typically this is done by alternating between ampicillin and kanamycin antibiotic markers, but this not only goes against the core thesis of the project but also doubles the exotic/regulated chemicals one would need to source to do this sort of iterative cloning themselves. The need for a second selection system is now critical. Enter citrate!
E. coli Citrate Use Cell Biology - EvoEd : EvoEd (evo-ed.org)
In the Citric Acid Cycle we all suffered to learn in our school days, citrate ions play a crucial role keeping our cells alive by cycling to replenish ATP, NADH, and FADH2 molecules via bond energy harvesting. This cycle is absolutely vital for the survival of many living things, but in E. coli can only import citrate from the environment in anaerobic conditions.
E. coli Citrate Use Cell Biology - EvoEd : EvoEd (evo-ed.org)
The way in which citrate comes into the E. coli cell is via the citT citrate/succinate antiporter. It trades a succinate ion (produced downstream in the Citric Acid cycle) with exogenous citrate and is the only way citrate can enter. The citT gene is expressed in anerobic conditions and repressed in the presence of oxygen. The idea I want to try is simple:
Express citT constitutively via a plasmid to maintain selection of cells carrying said plasmid on media where citrate is the sole carbon source. This has two likely constraints:
Citrate metabolism in aerobic conditions has been shown to spontaneously evolve (thanks Richard Lenski for your diligent 30+ year experiment).
Strongly expressing transmembrane proteins which hook into cell wall may be bad.