How do endoribonucleases (ERNs) work to decrease protein levels? Name 2 differences between how ERNs work and how proteases work.

Since protein level regulation is essential for the proper working of a cell, the system must have different levels at which to control it. Such is the case of ERNs and proteases. They both control said levels but from different ends of the process. ERNs degrade RNA sequences of different kinds depending on the enzyme (tRNA, mRNA, double or single-stranded…), thus regulating gene expression and the amount of protein that eventually gets produced.

Proteases, on the other hand, degrade peptides and amino acids directly, and can be part of the production or activation of new protein complexes. Furthermore, proteases are much more inespecific than endonuclease, that work by recognising specific sequences.

How does lipofectamine 3000 work? How does DNA get into human cells and how is it expressed?

Since eukaryotic cells cannot be transformed in the same way as prokaryotic ones, in order to introduce DNA, other systems need to be used. Vesicles, such as liposomes, can fuse to the membrane in order to enter, so that mechanism is hijacked to introduce DNA into the cells. Normally, however, external elements that enter the cell via vesicles are degraded as the vesicle turns into a lysosome. Lipofectamine 3000 works by stopping that process and helping the DNA getting to the nucleus.

Explain what poly-transfection is and why it’s useful when building neuromorphic circuits.

Poly-transfection is a method developed for the assessment of genetic systems in a way that is easier to prepare, while still having a wide enough coverage when compared to others like co-transfection.

Since biological systems are too complex for purely digital/binary computation, the need for more nuanced ways of measuring arose, in the form of neuromorphic/analog computing, which can measure in a continuous manner, rather than just in a binary one. The resulting complexes hold a lot of potential in furthering our understanding of biological systems, especially as it can start overlapping with AI development.

The complexity also means that establishing experimental conditions to test different elements in a circuit requires not just the absence/presence of a particular part, but rather a “balance” of sorts between the different parts.

This is what poly-transfection offers. Rather than transforming one single plasmid, or mixing two different plasmids with the transfection reagent, each plasmid is separately mixed with the reagent first, and then transfected along with the other species.

This allows for less preparations with a wide coverage, including cells with neither plasmid, either, or different amounts of both, thus increasing the amount of covered possibilities, and thus the range visible fluorescence.

Genetic Toggle Switches:

• Provide a detailed explanation of the mechanism behind genetic toggle switches, including how bi-stability is established and maintained.

  A toggle switch refers to any kind of switch that can exist in two distinct, stable states, but no intermediate ones. The easiest example of it is a light switch, having both “on” and ”off” states. In the context of genetics, it would refer to a system that would transcribe either one gene product or another in the presence of inducer molecules, but otherwise would produce neither.

  

  In this example, the fact that both promoters control the expression of each other’s repressor ensures bi-stability in the absence of any inducer. Once either inducer is introduced into the system, the expression levels of the system will switch to a monostable system, in which only one of the two products is transcribed.

• Describe at least one induction method used to switch states, including molecular signals or environmental factors involved.

  Gene induction can be regulated in multiple ways, and not always purely by specific molecules. While that is usually taken as the most common method of induction, there are many genes whose transcription is tied to environmental factors, such as specific temperatures or radiation. Even within the inducer/repressor molecules, we can separate within subsets. Some inducers bind to specific genetic regions, blocking their access to the transcription machinery. Others bind proteins whose function regulates gene expression. Alternatively, we have the case of riboswitches, which change their spatial conformation in the presence of a specific molecule, allowing for changes in gene expression to take place.

• Are there any limitations? How many ‘switches’ can we potentially chain? Is there a metabolic cost?

  In theory, toggle systems such as these shouldn’t have necessarily a limit of potential switches, as long as the different regulatory elements have similar transcription rates. However, the increase in switches imply a progressive increase the complexity of the system, assuming each repressor is regulated by a different promoter. The possibility of different repressors being induced by the same molecule only increases the number of possible states of the system. A system with n promoters would always require a repressor number ≤ n, with a number of inducers ≤ n, but since all of those different elements would have to be constantly transcribed at the required levels to hold everything in balance, both the complexity and the metabolic toll on the cells increase to probably untenable levels.

Natural Genetic Circuit Example:

• Identify and describe in detail a naturally occurring genetic circuit, emphasizing its biological function, components, and regulatory interactions.

  One of the earliest regulatory elements to be studied, the cI repressor of phage λ plays an essential role in maintaining a latent state, stopping the phage’s DNA from replicating, and the lysis and eventual release from the host from taking place. There are three main elements, the cI, and the left and right operators (OL and OR). Each of them contains three different sites that cI can bind to in different ways, looping the DNA sequence, and repressing lytic promoters pR and pL. Furthermore, under some conditions cI has a self-regulatory function, inhibiting its own transcription at certain levels.

  

Synthetic Genetic Circuit:

• Select and critically analyze a synthetic genetic circuit previously engineered by researchers (e.g., pDAWN). Provide details about its construction, components, intended function, and performance.

  For this, I initially considered discussing a system involving cI, as developed by Huang, Holtz and Maharbiz (2012) but finally I have selected the bistable toggle switch developed by Mishra, et al. (2021).

  This switch is intriguing not only because it works around protein phosphorylation, rather than the transcription or translation level; but also because of the much shorter scale of its time response, allowing the switch to respond within seconds. The system is based upon cross-repression of two different pairs of elements, with one input for each, a classic bi-stable system.

  They used both endogenous and exogenous proteins in the system:

  • HOG-MAPK pathway sensors SLN1 and SHO1
  • NOT gate downstream from SLN1
  • OR gate from them to MAPKK PBS2
  • MAPK HOG1
  • PRIME (phosphorylation-required interaction and mediated effect) system such that phosphorylation upstream means binding to and phosphorylating downstream proteins.
  • Chimeric proteins with 3 domains: ready-binder, phosphor-binder, and effector. Formed by fusing CRE1 or STAT5 to intramol his-kinase response regulator domains from SNL1, responsible for OR gates.
  • YPD1, allowing protein histidine K binding.
  • Chimeric protein produced by fusing HOG1, a protein tyr-phosphate from SHP-1, and GFP; and another by fusing HOT1 and transcription fact JAK2. Responsible for NOT gate.
  • Isopentenyl adenine (IP) and sorbitol as inducers.

• Discuss potential limitations or improvements suggested in subsequent literature or experimental data.

  One clear limitation of the system is the fact that it is engineered in yeast, which, while opening up the possibility of working with mammalian cells, fundamentally restricts the use in prokaryotes. Also, the need to create chimeric proteins in order to develop the system.

  However, approaches like this would facilitate the identification of similar regulatory pathways, and could eventually be used for other interactions beyond phosphorylation.

  There could even be coupling of these pathways to specific cellular functions, with the possibility of tissue-level responses. These toggle switches could also be used as an alternative to gene knockout strategies, allowing for a more controlled and tenable regulation.

  Furthermore, given the current increase in protein engineering research (directed evolution, structure prediction, AI…), the possibilities for using cells as medical or environmental sensors are widening.