From fcc2b1cdec2336960540c649e316804be19238c8 Mon Sep 17 00:00:00 2001
From: Haobin Zhu <robinzhb@stu.pku.edu.cn>
Date: Thu, 12 Oct 2023 14:39:23 +0000
Subject: [PATCH] Update 15.results.md

---
 content/15.results.md | 22 +++++++++++-----------
 1 file changed, 11 insertions(+), 11 deletions(-)

diff --git a/content/15.results.md b/content/15.results.md
index ab6f446..4b38a7c 100644
--- a/content/15.results.md
+++ b/content/15.results.md
@@ -14,7 +14,7 @@
 
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-*Escherichia coli* Nissle 1917 (EcN), as a probiotic, has tumor-targeting properties to some extent. So, our aim was to enhance this tendency, specifically targeting EcN to hypoxic tumor microenvironment (TME). Therefore, we focused on hypoxia-inducible promoters (HIPs), hoping to achieve explicit hypoxia targeting of EcN.
+*Escherichia coli* Nissle 1917 (EcN), as a probiotic, has tumour-targeting properties to some extent. So, our aim was to enhance this tendency by specifically targeting EcN to the hypoxic tumour microenvironment (TME). Therefore, we focused on hypoxia-inducible promoters (HIPs), hoping to achieve explicit hypoxia targeting of EcN.
 
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@@ -32,7 +32,7 @@ HIPs are regulated by the Fumarate and Nitrate Reduction regulator (FNR)[$^1$].
 
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-Thus, on the base of wild type PepT promoter, we strived to change the intensity of its expression by mutating TATA box, mutating FNR box, and changing the RBS. We use the PJUMP41-2A (sfGFP) plasmid (BBa_J428365, this plasmid referred below are abbreviated as O12, as it corresponds to the O12 well on the biobrick) as our vector This plasmid is characterized as a low-copy plasmid with spectinomycin resistance and carries the BBa_J23100 promoter along with the sfGFP sequence. Through reverse PCR and gibson assembly (details are shown in “Engineering”), we successfully replaced the J23100 promoter and RBS with the following 10 promoters + RBS (see **BBa_K4713116 - BBa_K4713125**):
+Thus, on the base of wild type PepT promoter, we strived to change the intensity of its expression by mutating TATA box, mutating FNR box, and changing the RBS. We used the PJUMP41-2A (sfGFP) plasmid (BBa_J428365, this plasmid referred below are abbreviated as O12, as it corresponds to the O12 well on the biobrick) as our vector This plasmid is characterized as a low-copy plasmid with spectinomycin resistance and carries the BBa_J23100 promoter along with the sfGFP sequence. Through reverse PCR and Gibson assembly (details are shown in “Engineering”), we successfully replaced the J23100 promoter and RBS with the following 10 promoters + RBS (see **BBa_K4713116 - BBa_K4713125**):
 
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@@ -142,7 +142,7 @@ We demonstrated the viability of the microfluidic chip by verifying the presence
 
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-Unfortunately, we didn't had enough time to apply our microfluidic chip on formal measurement. However, we believe that microscopic result will be similar to the macroscopic result.
+Unfortunately, we didn't have enough time to apply our microfluidic chip on formal measurement. However, we believe that microscopic result will be similar to the macroscopic result.
 
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@@ -150,7 +150,7 @@ Unfortunately, we didn't had enough time to apply our microfluidic chip on forma
 
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-In order to reduce the escape rate and improve targeting to the tumor microenvironment, our initial idea was to build an AND-gate system to sense hypoxia and high lactate simultaneously. **Fig. 6a** shows one of the traditional AND-gate systems[$^5$]. This system involves two signal inputs (Ara and aTc), which produce protein sicA and invF respectively that can bind and act as a transcriptional activator to initiate the expression of downstream *rfp* gene. We initially wanted to imitate this AND-gate structure and replace the downstream output with the necessary gene (*asd*) for EcN. The *asd* gene expresses aspartate semialdehyde dehydrogenase, which is required for the biosynthesis of lysine, threonine and methionine. Deletion of *asd* causes bacterial cell wall rupture and death. Supplementation with diaminopimelic acid (DAP) can promote the growth of *asd* knockout strains. Notably, DAP cannot be produced or metabolized from the host cell environment, making it an ideal strategy to reduce escape rates. High lactate is sensed through the native *lldPRD* lactate operon of EcN[$^6$]. But we are worried that this AND-gate structure may bring a greater expression burden to EcN.
+In order to reduce the escape rate and improve targeting to the tumour microenvironment, our initial idea was to build an AND-gate system to sense hypoxia and high lactate simultaneously. **Fig. 6a** shows one of the traditional AND-gate systems[$^5$]. This system involves two signal inputs (Ara and aTc), which produce protein sicA and invF respectively that can bind and act as a transcriptional activator to initiate the expression of downstream *rfp* gene. We initially wanted to imitate this AND-gate structure and replace the downstream output with the necessary gene (*asd*) for EcN. The *asd* gene expresses aspartate semialdehyde dehydrogenase, which is required for the biosynthesis of lysine, threonine and methionine. Deletion of *asd* causes bacterial cell wall rupture and death. Supplementation with diaminopimelic acid (DAP) can promote the growth of *asd* knockout strains. Notably, DAP cannot be produced or metabolized from the host cell environment, making it an ideal strategy to reduce escape rates. High lactate is sensed through the native *lldPRD* lactate operon of EcN[$^6$]. But we are worried that this AND-gate structure may bring a greater expression burden to EcN.
 
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@@ -246,7 +246,7 @@ Unfortunately, due to time limits, we did not experiment with the effect of expr
 
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-After the construction of hybrid promoter, our engineered bacteria had the ability to colonize specifically in the tumor tissues. According to our plan, the next step we need to find a method that can detect the location of the presence of bacteria. Bourdeau *et al*[$^9$] genetically modified the vesicular protein from a photosynthetic autotrophic bacterium cyanobacterium *Anabaena flos-aquae* and transformed it into E. coli BL21(AI) to successfully express the vesicular protein, which could be detected by medical ultrasound probes. However, this article still has the shortcoming that the bacteria can only reach the tumor by *in situ* injection. The ability of engineered bacteria to self-localize tumors is necessary if this method is to be applied to clinical treatment.
+After the construction of hybrid promoter, our engineered bacteria had the ability to colonize specifically in the tumour tissues. According to our plan, the next step we need to find a method that can detect the location of the presence of bacteria. Bourdeau *et al*[$^9$] genetically modified the vesicular protein from a photosynthetic autotrophic bacterium cyanobacterium *Anabaena flos-aquae* and transformed it into E. coli BL21(AI) to successfully express the vesicular protein, which could be detected by medical ultrasound probes. However, this article still has the shortcoming that the bacteria can only reach the tumour by *in situ* injection. The ability of engineered bacteria to self-localize tumours is necessary if this method is to be applied to clinical treatment.
 
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@@ -260,7 +260,7 @@ After the construction of hybrid promoter, our engineered bacteria had the abili
 
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-Fortunately, the scheme proposed by the Peking iGEM team to target the tumor microenvironment by engineering bacteria can make up for this deficiency. We hypothesized that ARG could be introduced into the engineered bacteria containing and induce its expression vesicles. Since only the engineered bacteria colonized in the tumor tissue could survive, the signal would only be detected in the tumor tissue when detected by a medical ultrasound probe.
+Fortunately, the scheme proposed by the Peking iGEM team to target the tumour microenvironment by engineering bacteria can make up for this deficiency. We hypothesized that ARG could be introduced into the engineered bacteria containing and induce its expression vesicles. Since only the engineered bacteria colonized in the tumour tissue could survive, the signal would only be detected in the tumour tissue when detected by a medical ultrasound probe.
 
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@@ -268,7 +268,7 @@ To prove this idea, pET28a_T7-ARG1 plasmid (addgene #106473, purchased from Miao
 
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-After centrifuged for 2h at 300*g*, the result shown as **Fig. 11**. The results for other induction concentrations are similar and will not be shown here.
+After centrifuging for 2h at 300*g*, the results are shown as **Fig. 11**. The results for other induction concentrations are similar and will not be shown here.
 
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@@ -320,7 +320,7 @@ After confirming that the pET28a_T7-ARG1 plasmid was available, we managed to ex
 
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-Based on the above presentation, we successfully expressed vesicular proteins in EcN that ultrasound could detect. The pET28a_T5-ARG plasmid combined with the hybrid promoter is expected to obtain engineered bacteria that can specifically colonize tumors and be detected by ultrasound. The engineered bacteria are expected to be used as an alternative to PET-CT in the future to provide help for the systemic detection of metastatic lesions in advanced pancreatic cancer.
+Based on the above presentation, we successfully expressed vesicular proteins in EcN that ultrasound could detect. The pET28a_T5-ARG plasmid, combined with the hybrid promoter, is expected to obtain engineered bacteria that can specifically colonize tumours and be detected by ultrasound. The engineered bacteria are expected to be used as an alternative to PET-CT in the future to provide help for the systemic detection of metastatic lesions in advanced pancreatic cancer.
 
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@@ -328,8 +328,8 @@ Based on the above presentation, we successfully expressed vesicular proteins in
 
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-Though tumor-targeting and ultrasound-sensing has been achieved, controlled immunogenicity during in vivo delivery is still a problem. In order to avoid viremia as much as possible, we expect to mask epitopes of EcN before ultrasound imaging and achieve rapid clearance after diagnosis. Thus, we focused on engineering the capsular polysaccharide (CAP). CAP is a loose mucous substance located on the surface of the cell wall, protecting bacteria from environmental influences. It is worth mentioning that the thickness of CAP changes dynamically, and if not generated endogenously, its thickness will naturally become thinner. Harimoto *et al* [$^{11}$] have designed an inducible capsular polysaccharide (iCAP) system. They used the *kfiC* gene to regulate the expression of CAP. As an endogenous gene in EcN, *kfiC* encodes a glycotransferase essential in CAP biosynthesis, thus its expression determines the rate of CAP production, and then impacts the thickness of cellular outer layer.
-Based on the previous work, we designed the following circuit (**Fig. 16a**): *kfiC* under the control of ParaB (an arabinose-inducible promoter) would be transformed into EcN *∆asd ∆kfic*. Prior to delivery, the bacteria would be induced by arabinose to start kfiC expression, and then encapsulation from CAP production, preparing it for host immune evasion and tumor focus localization. As CAP gradually degrades and cellular outer layer becomes thinner over time, the remaining bacteria in blood would be rapidly cleared, thus reducing long-term inflammatory response and toxicity risks (**Fig. 16b**) [$^{11}$].
+Though tumour-targeting and ultrasound-sensing has been achieved, controlled immunogenicity during in vivo delivery is still a problem. In order to avoid viremia as much as possible, we expect to mask epitopes of EcN before ultrasound imaging and achieve rapid clearance after diagnosis. Thus, we focused on engineering the capsular polysaccharide (CAP). CAP is a loose mucous substance located on the surface of the cell wall, protecting bacteria from environmental influences. It is worth mentioning that the thickness of CAP changes dynamically, and if not generated endogenously, its thickness will naturally become thinner. Harimoto *et al* [$^{11}$] have designed an inducible capsular polysaccharide (iCAP) system. They used the *kfiC* gene to regulate the expression of CAP. As an endogenous gene in EcN, *kfiC* encodes a glycotransferase essential in CAP biosynthesis, thus its expression determines the rate of CAP production, and then impacts the thickness of cellular outer layer.
+Based on the previous work, we designed the following circuit (**Fig. 16a**): *kfiC* under the control of ParaB (an arabinose-inducible promoter) would be transformed into EcN *∆asd ∆kfic*. Prior to delivery, the bacteria would be induced by arabinose to start kfiC expression, and then encapsulation from CAP production, preparing it for host immune evasion and tumour focus localization. As CAP gradually degrades and cellular outer layer becomes thinner over time, the remaining bacteria in blood would be rapidly cleared, thus reducing long-term inflammatory response and toxicity risks (**Fig. 16b**) [$^{11}$].
 
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@@ -344,7 +344,7 @@ Based on the previous work, we designed the following circuit (**Fig. 16a**): *k
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 We tried to knockout the *kfiC* gene from the genome of EcN *∆asd* in the same way as the *asd* gene (see Result 2.1), yet without success. A possible cause may be that both *asd* and *kfiC* genes are essential to bacterial cell wall synthesis, thus the absence of both would be fatal to EcN. We propose to change the output necessary gene of hybrid promoter to *ThyA*[$^{12}$]. *ThyA* encodes thymidylate synthase in E. coli, which is required in nucleotide biosynthesis pathways, and does not interfere with cell wall synthesis.
-Nevertheless, we demonstrated the feasibility of our iCAP system through modelling. By changing the elimination rate [$k_e$] in our MC model (see **Dry Lab - Model**), we were able to simulate an enrichment to the Tumor compartment as well as rapid clearance in other compartments with suitable elimination rates determined by CAP thickness.
+Nevertheless, we demonstrated the feasibility of our iCAP system through modelling. By changing the elimination rate [$k_e$] in our MC model (see **Dry Lab - Model**), we were able to simulate an enrichment to the tumour compartment as well as rapid clearance in other compartments with suitable elimination rates determined by CAP thickness.
 This design can be utilized to optimize therapeutic bacteria administration to maximize curative effects and minimize side effects. 
 
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GitLab