Genetic circuits, synthetic networks and memory systems

[research555]

Today I focused on two different areas; using bacteria and diagnostic tools and different components of genetic circuits. I already knew about the paper based circuit design but figured id include it here for everyone to see. I will also include a long summary of how the cI-Cro system can be used in a biotic biosensor on the bottom of the page. I wrote that summary a while ago but I feel like its very relevant.

Genetic Circuits

Toehold Switches & Ribocomputers

Papers

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265554/ (Toehold switches)
• https://www.nature.com/articles/nature23271 (Ribocomputing)

How it works

• Toehold switches (such as the ones used in paper based synthetic gene circuits) are RNA molecules with secondary structures of hairpin loops
• They contain toehold regions which can base pair with complementary mRNA
• They also contain ribosome binding sites, start codon and a repressed gene which is only expressed when the hairpin loop unwinds
• The loop only unwinds when complementary mRNA is bound to the toehold region
• This complementary mRNA is termed trigger RNA
• When they meet, the switch RNA unwinds, ribosomes are able to bind to the switch and begin producing the reporter protein

  Applications

  Sequence detection

  Logic gates

  OR

• Two toeholds put together A & B in a system called Gate RNA
• If the first toehold is unwound, a ribosome is able to bind and manually unwind the second toehold (B) and translate the reporter protein
• If the second toehold is unwound, the same process can take place
• This is why its called an OR gate. Either trigger A or trigger B is enough to cause reporter protein synthesis

  AND

  Trigger RNA

• The trigger RNA for the AND gate is produced by splitting the complementary region into two parts and adding u and u’ regions which are complementary to each other to the two components respectively
• The u and u’ regions when in contact will base pair leaving an opposite T-like shape on the bottom composed of the RNA that is complementary to the target sequence
• Alone, they cannot cause unwinding but together they can hence the AND gate
• Trigger RNA is only able to unwind the lower portion of the switch
• The ribosome binding site remains in the loop but can bind weakly to the ribosome so translation can occur

  NOT

  Trigger RNA

• The trigger RNA for the NOT gate is produced by creating RNA with two toehold regions on each side of the sequence that is complementary to the target sequence labelled u and v
• The trigger can bind to the gate RNA as usual

  Deactivating RNA

• The deactivating RNA is designed to be complementary to the trigger RNA
• It starts by binding to either the u or v regions and displaces the trigger RNA from the gate RNA, halting translation

  Multiple gates

• You can put multiple AND or OR gates on the same trigger RNA
• They showed that it was still effective and worked well with 4 AND gates and 6 OR gates on the same trigger RNA
• You can also put multiple AND NOT and OR gates on a single trigger
• Regulation of CRISPR-Cas9 guides

Designing Toehold switches is hard

• Sequence constraints
• Toeholds must form correct secondary structure
• mRNA can form secondary structures too which can make it hard for the toeholds to bind to them
• It’s hard to predict a toeholds efficiency
• Much of the above is dependent on energy and whether the bound state is more favorable than the unbound state which is hard to predict

Paper based synthetic networks

Paper

• https://www.sciencedirect.com/science/article/pii/S0092867414012914

Main outline

• Application of above papers
• Basically theres trigger RNA in the paper with toehold switches
• You then put your solution with the foreign mRNA like zika onto the paper
• It will then change color if zika is detected

In vivo diagnostics

Paper

• https://www.nature.com/articles/nbt.3879

One sentence summary

• Designing a trigger circuit that responds to tetrathionate (marker of inflammation) and they aimed to develop a strain that could detect this inflammatory marker and study the ability of the strain to colonize and function in the mouse gut over long periods of time

  Main outline

• Tetrathionate is detected by TtrR/TtrS two component system
• In the presence of tetrathionate, TtrS phosphorylates TtrR which activates expression via PttrBCA promoter.
• Downstream of this promoter they placed a cro gene and used the memory element they used before
• Their system showed that the engineered bacteria senses tetrathionate with low level inflammation not detected by histological analysis
• The bacteria was able to live for over 6 months in the gut without getting mutated or outcompeted by the natural murine gut flora

Programmable bacteria detect and record an environmental signal in the mammalian gut

Paper

• https://www.pnas.org/content/111/13/4838

Quick overview

• Proof of concept that E. coli can be engineered with a synthetic memory system based on the lambda phase cI/Cro genetic switch can sense and record antibiotic exposure during passage through the mouse gut.
• Hoping for this to be used in the future as a way for bacteria to be used as biosensors and effectors

  Rules

• Initial non-memory state should be highly stable and only fail due to mutation
• Memory state should be highly stable
• Engineered elements should be integrated into chromosomes rather than into plasmids to limit potential for loss
• Engineered elements should not impose a large fitness cost to the organism to prevent removal through natural selection

Main outline

• Composed of two elements

  Transcriptional memory element

• This element will be exhibit bistability and thus have on and off switch
• They used the cI/Cro lambda phage system due to its natural stability prior to induction (activation)
• This system only fails if theres mutations

  Trigger element

• The trigger element will cause the memory element to either switch from producing cro (which represses cI) or from producing cI (which represses cro)
• As a trigger they used ATC, which binds to tetR to cause tetracycline resistance, but downstream they placed a cro gene
• If ATC is detected, cro is produced (and lacZ) and cI is repressed. If not, cI is produced and cro is repressed and no lacZ is produced
• Starts off in the cI state and switches to cro on derepression via ATC

Conclusions

• Results were much better in engineered native gut bacteria rather than not
• Genetic circuits can be designed in laboratory strains then transferred onto native ones when you wanna actually use it
• There were no fitness cost to having the system

[SarahHagg]

Haotian (finished his PhD at the CRI a few months ago) was also working on riboregulators:

'I summarized a strategy towards universal design of riboregulators, termed “anti-flanking mechanism”, and concluded why state-of-art designs of synthetic riboregulators, represented by toehold switches, are not universal enough to meet a variety of requirements'. He used 'de-novo-designed RNA repressors of CRISPR RNA processing, termed anti-CRISPR RNAs (acrRNAs).'