Update results.md

docs/project/results.md

@@ -2,7 +2,7 @@
 
 ## Selection of of the E3 ligase
 
-After evaluating several well-known human E3 ligases that could be engineered, we decided to work on evolving SIAH1 and SIAH2 (collectively referred to as SIAH1/2 in this text), which are primarily associated with cellular stress response, such as hypoxia [^E3_1][^E3_2]. Both belong to the RING family of E3 ligases and share 86% sequence identity with nearly identical substrate-binding domains[^E3_3]. Their small sizes—282 amino acids for SIAH1 and 324 for SIAH2—are optimal for phage-assisted continuous evolution (PACE), as this allows for efficient expression in E. coli and packaging into M13 phages. Moreover, this small size compared to other E3 ligases also reduces the theoretical library size which is usually advantageous in directed evolution experiments. In general, neither SIAH1 nor SIAH2 are individually characterised to an extent we would like them to be but together they generate a full picture. SIAH2 has already been used successfully in _E. coli_ ubiquitination assays without any partner proteins except E1 and E2, suggesting that additional regulatory proteins aren’t required for its ubiquitination activity [^E3_4]. This streamlines the evolution processes by reducing potential points of failure in the selection system and keeps the plasmid sizes within an acceptable range. Given how similar SIAH1 is to SIAH2 in structure and function, we anticipate it will behave similarly in _E. coli_ as well. While these studies are missing for SIAH1, the binding of SIAH1 to specific peptide sequences has been well-studied via X-ray crystallography [^E3_5][^E3_6], which is missing for SIAH2. Together, the data available for SIAH1/2 offer a solid foundation for targeting new sequences and evolving either protein to recognize non-canonical substrates.
+After evaluating several well-known human E3 ligases that could be engineered, we decided to work on evolving SIAH1 and SIAH2 (collectively referred to as SIAH1/2 in this text), which are primarily associated with cellular stress response, such as hypoxia [^E3_1][^E3_2]. Both belong to the RING family of E3 ligases and share 86% sequence identity with nearly identical substrate-binding domains[^E3_3]. Their small sizes—282 amino acids for SIAH1 and 324 for SIAH2—are optimal for phage-assisted continuous evolution (PACE), as this allows for efficient expression in E. coli and packaging into M13 bacteriophages. Moreover, this small size compared to other E3 ligases also reduces the theoretical library size which is usually advantageous in directed evolution experiments. In general, neither SIAH1 nor SIAH2 are individually characterised to the extent we would like them to be but together they generate a full picture. SIAH2 has already been used successfully in _E. coli_ ubiquitination assays without any partner proteins except E1 and E2, suggesting that additional regulatory proteins aren’t required for its ubiquitination activity [^E3_4]. This streamlines the evolution processes by reducing potential points of failure in the selection system and keeps the plasmid sizes within an acceptable range. Given how similar SIAH1 is to SIAH2 in structure and function, we anticipate it will behave similarly in _E. coli_ as well. While these studies are missing for SIAH1, the binding of SIAH1 to specific peptide sequences has been well-studied via X-ray crystallography [^E3_5][^E3_6], which is missing for SIAH2. Together, the data available for SIAH1/2 offer a solid foundation for targeting new sequences and evolving either protein to recognize non-canonical substrates.
 
 The SIAH family recognizes its targets through a PXAXVXP degron motif [^E3_7], where conserved residues Pro, Ala, Val, and Pro face the SIAH binding pocket (Figure 1). Specificity is mainly determined by the Ala and Val residues in positions 3 and 5, as these pockets are too small to accommodate larger side chains [^E3_8]. Additionally, the Pro residue at position 7 interacts with Trp178 in SIAH, contributing further specificity. Among canonical substrates, the VXP sequence is highly conserved [^E3_6][^E3_9], while other residues show more variability, making them prime candidates to alter SIAH1/2 specificity for these positions.
 
@@ -32,9 +32,9 @@ Based on these five criteria, we searched published ubiquitination databases (10
 
 *For EGLN1 accurate degron was not identified, though two VXP motifs exist of which at least one should act as SIAH1/2 degron.
 
-ɑ-Synuclein is well known for causing Parkinson's disease (PD) and multiple systems atrophy (MSA). Therefore, optimization of SIAH1/2 binding to ɑ-Synuclein could be an attractive alternative evolution strategy in case reprogramming SIAH1/2 specificity  does not work as planned.
+ɑ-Synuclein is well known for causing Parkinson's disease (PD) and multiple systems atrophy (MSA). Therefore, optimization of SIAH1/2 binding to ɑ-Synuclein could be an attractive alternative evolution strategy in case reprogramming SIAH1/2 specificity does not work as planned.
 
-NLRP3 was selected as an evolutionary target, for its mentioned clinical relevance in hepatic  and neurodegenerative diseases. Furthermore, NLRP3 is an attractive target as it contains two VXP motifs (at positions 200 and 707), located in disordered regions on the surface of NLRP3 (Figure 2). This motif seems to be highly conserved among canonical SIAH1/2 substrates. Therefore, we assume our target choice is limited to proteins naturally displaying this motif on its surface, while other residues involved in binding could be changed. This would allow an evolution of SIAH1/2 to adapt for recognizing different VXP surrounding residues, while retaining the conserved VXP binding motif. Furthermore, lysins are found within the VXP surrounding residues, which are needed for ubiquitination. Remarkably, one of these lysins (K689) is natively polyubiquitinated and leads to the canonical degradation of NLRP3 [^substr_1], hence suggesting that polyubiquitination of K689 is a viable strategy of inducing NLRP3 degradation. With 1036 amino acids and its potential for oligomerization, NLRP3 could constrain our evolutionary system. Thus two peptide fragments of NLRP3 containing the VXP motif as well as surrounding residues were incorporated in the selection system.
+NLRP3 was selected as an evolutionary target, for its mentioned clinical relevance in hepatic and neurodegenerative diseases. Furthermore, NLRP3 is an attractive target as it contains two VXP motifs (at positions 200 and 707), located in disordered regions on the surface of NLRP3 (Figure 2). This motif seems to be highly conserved among canonical SIAH1/2 substrates. Therefore, we assume our target choice is limited to proteins naturally displaying this motif on its surface, while other residues involved in binding could be changed. This would allow an evolution of SIAH1/2 to adapt for recognizing different VXP surrounding residues while retaining the conserved VXP binding motif. Furthermore, lysins are found within the VXP surrounding residues, which are needed for ubiquitination. Remarkably, one of these lysins (K689) is natively polyubiquitinated and leads to the canonical degradation of NLRP3 [^substr_1], hence suggesting that polyubiquitination of K689 is a viable strategy for inducing NLRP3 degradation. With 1036 amino acids and its potential for oligomerization, NLRP3 could constrain our evolutionary system. Thus two peptide fragments of NLRP3 containing the VXP motif as well as surrounding residues were incorporated in the selection system.
 
 <figure markdown>
 ![Figure_nlrp3](https://idec-teams.github.io/2024_Evolution_Suisse/img/result figures/NLRP3_figure.png)
@@ -102,7 +102,7 @@ We were able to show that phage propagation is dependent on both the substrate o
 We suspected that the background phage propagation we observed could be caused by one of two things: 
 
 1. Leaky transcription of _gIII_: the gene controlling phage growth is turned on without the split-RNAP components being present. 
-2. Spontaneous assembly of the split-RNAP subunits: The two halves of the RNAP are coming together on their own, without the need of ubiquitination of the target substrate.
+2. Spontaneous assembly of the split-RNAP subunits: The two halves of the RNAP are coming together on their own, without the need for ubiquitination of the target substrate.
 
 Both hypotheses lead to _gIII_ expression independent of the E3 ligase activity. To test these hypotheses, we quantified phage propagation in cells that had only one half of the RNAP. In these cells, phage propagation was suppressed, confirming that both halves of the RNAP are required to activate _gIII_ expression. This means that accidental activation of _gIII_ wasn’t the issue. Instead, these results show that the two enzyme halves were probably joining together on their own, causing phage propagation without the involvement of the E3 ligase.
 
@@ -111,14 +111,14 @@ Both hypotheses lead to _gIII_ expression independent of the E3 ligase activity.
 <figcaption> Figure 8: Phage propagation in the presence and absence of split-RNAP components. This figure shows how much the phage population changed after overnight incubation with bacteria missing one of the two halves of the split-RNAP. The results are shown as a log2-fold change, meaning the numbers show how much the phage count increased or decreased, compared to the starting level. NC (negative control) is a bacterial strain lacking both parts of the system. PC (positive control) is a bacterial strain with the full system.</figcaption>
 </figure>
 
-### How can we reduce unwanted, E3-ligase independent page propagation? 
+### How can we reduce unwanted, E3-ligase-independent phage propagation? 
 We observed that the RNAP enzyme can assemble and become active before the phages even enter the bacterial cells. This early assembly can lead to unwanted phage propagation, which interferes with our goal to link phage propagation to E3 ligase activity. To overcome this problem, we changed the way we control the production of the N- and C-terminal halves of the RNAP. Instead of using a constitutive promoter, which is always active and produces the protein, we switched to a promoter that can be turned on when needed. For this, we chose two different inducible systems. One is switched on by adding vanillic acid (pVan promoter), the other by the stress response caused by phage infection (phage shock promoter). These new systems let us delay the production of the RNAP halves until just before or during phage infection. By regulating when these components are expressed, we can reduce the unintended assembly of the RNAP. This method can easily be added to the PACE system, allowing precise control of the timing of RNAP production. Experiments with this improved system are currently being performed.
 
 ### Enhancing SIAH1 genetic diversity using drift. 
 
 During PACE, both phages and infected bacteria are continuously removed from the lagoon. If phages propagate too slowly there's a risk that all the phages could be washed out before they can replicate and evolve. This is especially risky at the start of the experiment, when the initial activity of the E3 ligase may be too low to maintain sufficient phage propagation. To mitigate this risk, a process called "drift" is used before the evolution starts. Drift allows random mutations to occur without any selection pressure, increasing the genetic diversity among the phages. This genetic variability helps prevent phage washout by giving the population a better chance of harbouring variants with higher activity that can sustain propagation in the lagoon.
 
-To this end, we propagated the SIAH1 SP phages in bacterial cells that contain a special drift plasmid (DP6). The DP6 plasmid not only increases the mutation rate but also supplies the necessary pIII protein, allowing phages to replicate regardless of the E3 ligase's activity. Over eight infection and growth cycles (called passages), we sequenced the SIAH1 SP phages, and estimated the amount of mutations at different positions in the SIAH1 gene. With each passage, we saw the number of mutated phages increase, creating a highly diverse pool of SIAH1 SP phages. This pool can then serve as a robust starting pool for the next stages of evolutionary experiments, reducing the risk of phage washout and enhancing the chances of evolving phages with improved E3 ligase activity.
+To this end, we propagated the SIAH1 SP phages in bacterial cells that contain a special drift plasmid (DP6). The DP6 plasmid not only increases the mutation rate but also supplies the necessary pIII protein, allowing phages to replicate regardless of the E3 ligase's activity. Over eight infection and growth cycles (called passages), we sequenced the SIAH1 SP phages and estimated the amount of mutations at different positions in the SIAH1 gene. With each passage, we saw the number of mutated phages increase, creating a highly diverse pool of SIAH1 SP phages. This pool can then serve as a robust starting pool for the next stages of evolutionary experiments, reducing the risk of phage washout and enhancing the chances of evolving phages with improved E3 ligase activity.
 
 <figure markdown>
 ![Figure_SIAH1_drift](https://idec-teams.github.io/2024_Evolution_Suisse/img/result figures/Boxplot_quality_positions_wiki.png)