@@ -162,7 +162,7 @@ export function Engineering() {
<h4>Test</h4>
<p>After co-culture with different concentraions of PEA for different time (0,4,8,12 and 24h), the fluorescence was measured on microplate reader by excitation at 410 nm and detection of emission at 500 nm. OD600 (absorbance of 600nm) was also measured on microplate reader for normalization.</p>
<h4>Learn</h4>
<p>Results showed a significant increase in fluorescent intensity along with the increased level of PEA concentration. 100 ng/ml PEA presented the best capability of induction, and showed significant difference compared with other concentrations of PEA starting from 12 hours.</p>
<p>Results showed a significant increase in fluorescent intensity along with the increased level of PEA concentration. 100 ng/ml PEA presented the best capability of induction, and showed significant difference compared with other concentrations of PEA starting from 12 hours(Figure 6).</p>
@@ -187,9 +187,9 @@ export function Engineering() {
<p>The pathogenesis of HE is complicated, the mainstream theories include Ammonia Toxicity Theory and False Neurotransmitter Theory. Apart from endeavoring to metabolize ammonia (see details in our Design page) , we also put insights into reducing false neurotransmitters in our preliminary designs.</p>
<p>A false neurotransmitter is a chemical substance that closely resembles and mimics the function of a neurotransmitter in the nervous system. Examples include 5-MeO-αMT, which mimics serotonin, and α-methyldopa.These chemicals can be accumulated by a neuron or secretory cell, are then packaged in secretory / synaptic vesicles, and then released with other neurotransmitters when an action potential provides the necessary stimulus for release[8]. Patients who develop liver failure can not digest those aromatic amino acids properly, and this can lead to false neuro-transmitters accumulation, causing neuro system symptoms.</p>
<h5>TPH1: metabolize Trp</h5>
<p>Tryptophan, through different metabolic pathways, can be converted into kynurenine, serotonin, and indole[9]. Tryptophan hydroxylase 1 (TPH1), enabling the conversion of tryptophan (Trp) into serotonin (5-HT) and reducing excess aromatic amino acids. Since serotonin synthesized in the gut cannot cross the blood-brain barrier or affect central nervous system function, there is no concern about adverse effects on the central nervous system. Therefore, we designed a plasmid to express TPH1 as our metabolic module(Figure 8a).</p>
<p>Tryptophan, through different metabolic pathways, can be converted into kynurenine, serotonin, and indole[9]. Tryptophan hydroxylase 1 (TPH1), enabling the conversion of tryptophan (Trp) into serotonin (5-HT) and reducing excess aromatic amino acids. Since serotonin synthesized in the gut cannot cross the blood-brain barrier or affect central nervous system function, there is no concern about adverse effects on the central nervous system. Therefore, we designed a plasmid to express TPH1 as our metabolic module(Figure 7a).</p>
<h5>MAOB: metabolize PEA</h5>
<p>As we determined PEA to be the sensory substance in the sensing module, we also did literature reading on how it could be metabolized in the human body: by monoamine oxidase B (MAOB). Therefore, we designed a plasmid to heterologously express MAOB of Homo Sapiens in E.coli (Figure 8b).</p>
<p>As we determined PEA to be the sensory substance in the sensing module, we also did literature reading on how it could be metabolized in the human body: by monoamine oxidase B (MAOB). Therefore, we designed a plasmid to heterologously express MAOB of Homo Sapiens in E.coli (Figure 7b).</p>
@@ -209,14 +209,14 @@ export function Engineering() {
<p>To demonstrate the efficiency of PEA degradation,we transformed BL21 with plasmid Ptac-RBS-MAOB and cocultured the engineered bacteria with 0, 10, 25, 50 and 100 ng/ml PEA for 16 hours. Then we collected the culture medium and filtered through 0.22μM PTFE membrane. PEA concentration was measured via HPLC. The results showed a decrease in PEA concentration in all groups, including the control group transformed with pET28a(+) vector (Table 1). Data shows mean,n=3 independent experiments. Unfortunately, the results couldn't verify the function of MAOB, we suspect the intrinsic TynA enzyme in E.coli may mediate the degradation of PEA according to literature[10].</p>
<h4>Learn</h4>
<p>The unsuccessful attempts to express TPH1 and negative results of PEA degradation made us focus on metabolizing ammonia by expressing the GS enzyme (see details in our Design page). Fortunately, we successfully validate the feasibility of GS as our metabolic module and assembled it with sensing module (see details in our Result Page) . The final engineered design is shown in Figure 9.</p>
<p>The unsuccessful attempts to express TPH1 and negative results of PEA degradation made us focus on metabolizing ammonia by expressing the GS enzyme (see details in the Design section of our Description Page). Fortunately, we successfully validate the feasibility of GS as our metabolic module and assembled it with sensing module (see details in our Result Page) . The final engineered design is shown in Figure 8.</p>
<p>[1]ZHOU Yi-kang,WU Yi-nan,WANG Tian-min, ZHENG Xiang,XING Xin-hui,ZHANG Chong.Metabolite Biosensor：A Useful Synthetic Biology Tool to Assist the Construction of Microbial Cell Factory[J]. Biotechnology Bulletin, 2017, 33(1): 1-11.
[2]Xun, Z., Yao, X. & Ou, Q. Emerging roles of bile acids in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Cell Mol Immunol 20, 1087–1089 (2023).
[3]Liu N, Feng J, Lv Y, Liu Q, Deng J, Xia Y, Guo C, Zhou Y. Role of bile acids in the diagnosis and progression of liver cirrhosis: A prospective observational study. Exp Ther Med. 2019 Nov;18(5):4058-4066.
[4]Martínez-Fernández JA, Bravo D, Peirotén Á, Arqués JL, Landete JM. Bile-induced promoters for gene expression in Lactobacillus strains. Appl Microbiol Biotechnol. 2019 May;103(9):3819-3827.
[5]Wang, B., Kitney, R., Joly, N. et al. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat Commun 2, 508 (2011).
[6]Rottinghaus AG, Xi C, Amrofell MB, Yi H, Moon TS. Engineering ligand-specific biosensors for aromaticamino acids and neurochemicals. Cell Syst. 2022 Mar 16;13(3):204-214.e4.
[7]Nardelli S, Gioia S, Faccioli J, Riggio O, Ridola L. Hepatic encephalopathy - recent advances in treatment and diagnosis. Expert Rev Gastroenterol Hepatol. 2023 Mar;17(3):225-235.
[8]Abarca Monge, Sergio. “Cambio climático y plagas en el trópico.” Alcances Tecnológicos (2018): n. pag. doi:10.35486/at.v12i1.40.g27
[9]Xue C, Li G, Zheng Q, Gu X, Shi Q, Su Y, Chu Q, Yuan X, Bao Z, Lu J, Li L. Tryptophan metabolism in health and disease. Cell Metab. 2023 Aug 8;35(8):1304-1326. doi: 10.1016/j.cmet.2023.06.004. Epub 2023 Jun 22. PMID: 37352864.
[10]Zeng J, Spiro S. Finely tuned regulation of the aromatic amine degradation pathway in Escherichia coli. J Bacteriol. 2013 Nov;195(22):5141-50. doi: 10.1128/JB.00837-13. </p>
<p>[1]ZHOU Yi-kang,WU Yi-nan,WANG Tian-min, ZHENG Xiang,XING Xin-hui,ZHANG Chong.Metabolite Biosensor：A Useful Synthetic Biology Tool to Assist the Construction of Microbial Cell Factory[J]. Biotechnology Bulletin, 2017, 33(1): 1-11.</p>
<p>[2]Xun, Z., Yao, X. & Ou, Q. Emerging roles of bile acids in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Cell Mol Immunol 20, 1087–1089 (2023).</p>
<p>[3]Liu N, Feng J, Lv Y, Liu Q, Deng J, Xia Y, Guo C, Zhou Y. Role of bile acids in the diagnosis and progression of liver cirrhosis: A prospective observational study. Exp Ther Med. 2019 Nov;18(5):4058-4066.</p>
<p>[4]Martínez-Fernández JA, Bravo D, Peirotén Á, Arqués JL, Landete JM. Bile-induced promoters for gene expression in Lactobacillus strains. Appl Microbiol Biotechnol. 2019 May;103(9):3819-3827.</p>
<p>[5]Wang, B., Kitney, R., Joly, N. et al. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat Commun 2, 508 (2011). </p>
<p>[6]Rottinghaus AG, Xi C, Amrofell MB, Yi H, Moon TS. Engineering ligand-specific biosensors for aromaticamino acids and neurochemicals. Cell Syst. 2022 Mar 16;13(3):204-214.e4.</p>
<p>[7]Nardelli S, Gioia S, Faccioli J, Riggio O, Ridola L. Hepatic encephalopathy - recent advances in treatment and diagnosis. Expert Rev Gastroenterol Hepatol. 2023 Mar;17(3):225-235.</p>
<p>[8]Abarca Monge, Sergio. “Cambio climático y plagas en el trópico.” Alcances Tecnológicos (2018): n. pag. doi:10.35486/at.v12i1.40.g27</p>
<p>[9]Xue C, Li G, Zheng Q, Gu X, Shi Q, Su Y, Chu Q, Yuan X, Bao Z, Lu J, Li L. Tryptophan metabolism in health and disease. Cell Metab. 2023 Aug 8;35(8):1304-1326.</p>
<p>[10]Zeng J, Spiro S. Finely tuned regulation of the aromatic amine degradation pathway in Escherichia coli. J Bacteriol. 2013 Nov;195(22):5141-50. </p>
