From 954f70568c7cea659373c7e8bb274c164004285c Mon Sep 17 00:00:00 2001 From: Haobin Zhu <robinzhb@stu.pku.edu.cn> Date: Thu, 12 Oct 2023 14:23:43 +0000 Subject: [PATCH] Update 14.design.md --- content/14.design.md | 15 +++++++-------- 1 file changed, 7 insertions(+), 8 deletions(-) diff --git a/content/14.design.md b/content/14.design.md index d98ed31..f26942e 100644 --- a/content/14.design.md +++ b/content/14.design.md @@ -1,4 +1,3 @@ - <center> # **Design** @@ -12,7 +11,7 @@ <br> -In order to realize tumour-targeting and drug delivery in EcN, Peking 2023 designed three genetic circuits. +To achieve tumour-targeting and drug delivery in EcN, Peking 2023 has designed three genetic circuits. <br> @@ -28,7 +27,7 @@ Considering the strain has to express multiple additional proteins and carry exo <br> -A pivotal aspect of our innovative project involves constructing a hybrid promotor. TME, characterized by hypoxia and elevated lactate levels, remains relatively stable despite genetic mutations9. Thus, we establish an AND-gate-equivalent part, driving the expression of a knock-out essential gene (*asd*) on a loop vector <b><font color=#8B0012>Fig.1</font></b>. Consequently, the engineered *Escherichia coli* strain Nissle 1917 (or EcN, a safe probiotic strain widely used in the clinic) can only thrive and multiply in a hypoxia and lactate-abundant environment, achieving precise targeting of pancreatic cancer. +A pivotal aspect of our innovative project involves constructing a hybrid promoter. TME, characterized by hypoxia and elevated lactate levels, remains relatively stable despite genetic mutations. Thus, we establish an AND-gate-equivalent part, driving the expression of a knock-out essential gene (*asd*) on a loop vector <b><font color=#8B0012>Fig.1</font></b>. Consequently, the engineered *Escherichia coli* strain Nissle 1917 (or EcN, a safe probiotic strain widely used in the clinic) can only thrive and multiply in a hypoxia and lactate-abundant environment, achieving precise targeting of pancreatic cancer. <br> @@ -48,7 +47,7 @@ A pivotal aspect of our innovative project involves constructing a hybrid promot <br> -In *Escherichia coli* and *Salmonella typhimurium*, the Fumarate and Nitrate Reduction regulator (FNR) is a key regulator of hypoxia-induced promoters. Under hypoxic conditions, FNR holds its grasp on an oxygen-labile [4Fe-4S] cluster, initiating its own homodimerization and binding to a specific DNA site termed “FNR boxâ€, located in -35 region. Upon its binding, the homodimer interacts with the RNA polymerase binding to TATA box (with a conserved sequence of TATAAT) at -10 region, thus activating transcription <b><font color=#8B0012>Fig.2</font></b>. +In *Escherichia coli* and *Salmonella typhimurium*, the Fumarate and Nitrate Reduction regulator (FNR) is a key regulator of hypoxia-induced promoters. Under hypoxic conditions, FNR maintains its hold on an oxygen-labile [4Fe-4S] cluster, triggering its own homodimerization and binding to a specific DNA site termed “FNR boxâ€, located in -35 region. Upon its binding, the homodimer interacts with the RNA polymerase binding to TATA box (with a conserved sequence of TATAAT) at -10 region, thus activating transcription <b><font color=#8B0012>Fig.2</font></b>. <br> @@ -71,7 +70,7 @@ In *Escherichia coli* and *Salmonella typhimurium*, the Fumarate and Nitrate Red <br> -The LldPRD operon in Escherichia coli mediates aerobic L-lactate metabolism. This operon comprises three genes organized into a single transcriptional unit, inducible by L-lactate. Specifically, LldD codes for the dehydrogenase, LldP for the permease facilitating L-lactate uptake in vivo, and LldR for a regulatory protein. Two LldR binding sites exist within the operon: one at -90 and another at +40. LldR dimers form at these sites, potentially repressing the promoter through DNA loop formation, which inhibits RNA polymerase binding. Lactate can alleviate this repression. At high L-lactate concentrations, the LldR dimer disassociates, permitting transcription. <b><font color=#8B0012>Fig.3</font></b> +The LldPRD operon in Escherichia coli mediates aerobic L-lactate metabolism. This operon comprises three genes organized into a single transcriptional unit, inducible by L-lactate. Specifically, LldD codes for the dehydrogenase, LldP for the permease facilitating L-lactate uptake in vivo, and LldR for a regulatory protein. The operon contains two LldR binding sites: one at -90 and another at +40. LldR dimers form at these sites, potentially repressing the promoter through DNA loop formation, which inhibits RNA polymerase binding. Lactate can alleviate this repression. At high L-lactate concentrations, the LldR dimer disassociates, permitting transcription. <b><font color=#8B0012>Fig.3</font></b> <br> @@ -95,7 +94,7 @@ The LldPRD operon in Escherichia coli mediates aerobic L-lactate metabolism. Thi <br> -After investigation, we found it theoretically possible to integrate two promoters in one sequence. As pLldR functions by forming steric hindrance while pPepT functions by FNR combining. We can replace the constitutive promoter in BBa_K1847008 with pPepT. Thus, the promoter can function like an AND-gate while not producing additional peptides.<b><font color=#8B0012>Fig.4</font></b> +After investigation, we found it theoretically possible to integrate two promoters in one sequence. pLldR functions by forming steric hindrance, whereas pPepT operates through the combination of FNR. We can replace the constitutive promoter in BBa_K1847008 with pPepT. Thus, the promoter can function like an AND-gate while not producing additional peptides.<b><font color=#8B0012>Fig.4</font></b> <br> @@ -118,7 +117,7 @@ After investigation, we found it theoretically possible to integrate two promote <br> -a paper published in Nature in 2018 (Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts, Raymond W. Bourdeau, Audrey Lee-Gosselin.) caught our attention. According to the report, the research team 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. They named this class of vesicles ARGs (Acoustic Reporter Genes). Such vesicular proteins can be detected by medical ultrasound probes. Therefore, the research team put forward the preliminary idea of using engineered bacteria to indicate tumour tissue. +A paper published in Nature in 2018 (Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts, Raymond W. Bourdeau, Audrey Lee-Gosselin.) caught our attention. According to the report, the research team 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. They named this class of vesicles ARGs (Acoustic Reporter Genes). Such vesicular proteins can be detected by medical ultrasound probes. Therefore, the research team put forward the preliminary idea of using engineered bacteria to indicate tumour tissue. <br> @@ -161,7 +160,7 @@ To achieve controlled immunogenicity of engineered bacteria with minimal harm to <font size=3 color=grey> -**Figure 6 | The schematic diagram of dynamic circuit17. By controlling the strength and duration of arabinose induction, we aim to achieve the desired effect as "iCAP" shown in the figure, while minimizing toxicity reactions in the organism as much as possible.** +**Figure 6 | The schematic diagram of dynamic circuit17. By regulating the intensity and duration of arabinose induction, we strive to replicate the desired effect as "iCAP" shown in the figure, with the goal of minimizing toxic reactions in the body.** </font> -- GitLab