Difference between revisions of "Part:BBa K3771000"
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<br><b style="font-size:1.3rem">Description</b>
 
<br><b style="font-size:1.3rem">Description</b>
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<p>JJU (cysteine lyase) is an enzyme weighing approximately 43.1 kDa. JJU catalyzes the beta replacement reaction of L-cysteine and sulfite to form L-cysteate and hydrogen sulfide[1].</p>
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<p>JJU (cysteine lyase) is an enzyme weighing approximately 43.1 kDa. JJU catalyzes the beta replacement reaction of L-cysteine and sulfite to form L-cysteate and hydrogen sulfide<a href="https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10" alt="" target="_blank">[1]</a>.</p>
  
 
<br><b style="font-size:1.3rem">Usage</b>
 
<br><b style="font-size:1.3rem">Usage</b>
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<p>The three taurine production pathways incorporated into our <i>E. coli</i> include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway[1,2].</p>
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<p>The three taurine production pathways incorporated into our <i>E. coli</i> include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway<a href="https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10" alt="" target="_blank">[1]</a>.</p>
  
  
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  <p align="center">Fig. 1. Taurine pathways in <i>E. coli</i> [1,2].</p>
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  <p align="center">Fig. 1. Taurine pathways in <i>E. coli</i> <a href="https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10" alt="" target="_blank">[1]</a>.</p>
  
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<p>JJU is an enzyme that is part of the JJU-CoaBC taurine production pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cysteine to L-cystate, which then becomes taurine by CoaBC[1]. </p>
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<p>JJU is an enzyme that is part of the JJU-CoaBC taurine production pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cysteine to L-cystate, which then becomes taurine by CoaBC<a href="https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10" alt="" target="_blank">[1]</a>. </p>
  
  
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   <p align="center">Fig. 2. Biobrick of <i>P<sub>trc</sub></i>-JJU</i>
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   <p align="center">Fig. 2. Biobrick of <i>P<sub>trc</sub>-jju</i>
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<p>JJU enzyme was used in <i>in vitro</i> testing of taurine production. The sequence for JJU enzyme and <i>trc</i> promoter (<a href="https://parts.igem.org/Part:BBa_K864400" alt="" target="_blank">BBa_K864400</a>) were ligated and transformed into <i>E. coli</i>. By adding L-cysteine substrate into a reaction solution, JJU can help catalyze the conversion of L-cysteine to taurine. Taurine concentrations were determined using high-performance liquid chromatography (HPLC).</p>  
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<p>JJU enzyme was used in <i>in vitro</i> test of taurine production. The sequence for JJU enzyme and <i>trc</i> promoter (<a href="https://parts.igem.org/Part:BBa_K864400" alt="" target="_blank">BBa_K864400</a>) were ligated and transformed into <i>E. coli</i>. By adding L-cysteine substrate into a reaction solution, JJU can help catalyze the conversion of L-cysteine to taurine. Taurine concentration were determined by high-performance liquid chromatography (HPLC).</p>  
 
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<br><b style="font-size:1.3rem">Characterization</b>
 
<br><b style="font-size:1.3rem">Characterization</b>
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  <p align="center">Fig. 3. Agarose gel electrophoresis results showing amplified <i>jju</i> gene fragment</p>
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  <p align="center">Fig. 3. Agarose gel electrophoresis result showing amplified <i>jju</i> gene fragment. M: Marker; Lane 1: jju (1180 bp)</p>
  
 
    
 
    
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<br>In our <i>in vitro</i> testing of taurine production by JJU, JJU was transformed into <i>E. coli</i> BL21(DE3) strain. Supernatant (S) and whole cell (WC) samples were collected to confirm extracellular and intracellular protein expression by SDS-PAGE. <br>
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<br>In our <i>in vitro</i> test of taurine production by JJU, JJU was transformed into <i>E. coli</i> BL21(DE3) strain. Soluable protein (S) and whole cell (WC) samples were collected to confirm extracellular and intracellular protein expression by SDS-PAGE. <br>
  
 
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  <p align="center">Fig. 4. Confirmation of JJU expression by SDS-PAGE</p>
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  <p align="center">Fig. 4. Confirmation of JJU expression by SDS-PAGE. M: Marker; Lane 1: whole cell of JJU in BL21(DE3); Lane 2: soluble protein of JJU in BL21(DE3) (~43 kDa);
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Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa)
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</p>
  
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<br>Whole cell and supernatant JJU and CoaBC samples were collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. High-performance liquid chromatography (HPLC) was conducted to determine taurine concentration. Because JJU concentrations were lower, two times as much JJU supernatant volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine. As shown in figure 2, 2:1 ratio of JJU to CoaBC supernatant volume had the highest taurine concentration of around 95 mg/L. <br>
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<br>Whole cell and soluable protein JJU and CoaBC samples were collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. High-performance liquid chromatography (HPLC) was conducted to determine taurine concentration. Because JJU concentration was lower, two times as much JJU soluable protein volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine. As shown in figure 5, 2:1 ratio of JJU to CoaBC soulable protein volume had the highest taurine concentration of around 95 mg/L. <br>
  
 
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   <img src="https://2021.igem.org/wiki/images/1/14/T--NCKU_Tainan--1.png" style="width:50%;">
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   <img src="https://2021.igem.org/File:T--NCKU_Tainan--picture_12.png" style="width:50%;">
 
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  <p align="center">Fig. 5. (<i>in vitro</i> 1 taurine)</p>
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  <p align="center">Fig. 5. Taurine production of both JJU and CoaBC in BL21(DE3) in whole cell samples and soluble protein</p>
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<br><b style="font-size:1.1rem">Taurine Production of JJU in <i>E. coli</i> BD7G strain</b>
 
<br><b style="font-size:1.1rem">Taurine Production of JJU in <i>E. coli</i> BD7G strain</b>
 
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<br>Since JJU expression in BL21(DE3) strain was not prominent in the supernatant, we performed another <i>in vitro</i> test in which <i>P<sub>T7</sub>-jju</i> was transformed into BD7G strain instead of the BL21(DE3) strain. The BD7G strain contains chaperone protein GroELS that aids in protein folding [1]. SDS-PAGE results confirm JJU expression in both supernatant and whole cell samples.<br>
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<br>Since JJU expression in BL21(DE3) strain was not prominent in the soluable protein, we performed another <i>in vitro</i> test in which <i>P<sub>T7</sub>-jju</i> was transformed into BD7G strain instead of the BL21(DE3) strain. The BD7G strain contains chaperone protein GroELS that aids in protein folding<a href="https://researchoutput.ncku.edu.tw/zh/publications/enhanced-5-aminolevulinic-acid-production-by-co-expression-of-cod" alt="" target="_blank">[2]</a>. SDS-PAGE results confirm JJU expression in both soluable protein and whole cell samples.<br>
  
 
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  <p align="center">Fig. 6. (PAGE <i>in vitro</i> 2)</p>
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  <p align="center">Fig. 6. Confirmation of JJU expression by SDS-PAGE. M: Marker; Lane 1: whole cell of JJU in BD7G; Lane 2: soluble protein of JJU in BD7G (~43 kDa);
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Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa)  
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</p>
  
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<br>The whole cell and supernatant JJU and CoaBC samples are collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. When <i>P<sub>T7</sub>-jju</i> was transformed into the BD7G strain, 1:1 ratio of JJU to CoaBC supernatant had the highest taurine production, as shown in figure 20. This suggests the activity level of JJU does not significantly differ from that of CoaBC, and both are equally crucial and effective in converting L-cysteine to taurine.<br>
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<br>The whole cell and soluable protein JJU and CoaBC samples are collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. When <i>P<sub>T7</sub>-jju</i> was transformed into the BD7G strain, 1:1 ratio of JJU to CoaBC supernatant had the highest taurine production, as shown in Fig. 7. This suggests the activity level of JJU does not significantly differ from that of CoaBC, and both are equally crucial and effective in converting L-cysteine to taurine.<br>
  
 
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   <img src="https://2021.igem.org/wiki/images/0/0d/T--NCKU_Tainan--2.png" style="width:50%;">
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  <p align="center">Fig. 7. (<i>in vitro</i> 2 taurine)</p>
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  <p align="center">Fig. 7. Taurine production of JJU in BD7G and CoaBC in BL21(DE3) in soluble protein samples.
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<br><b style="font-size:1.3rem">References</b>
 
<br><b style="font-size:1.3rem">References</b>

Revision as of 17:10, 21 October 2021


JJU


Description

JJU (cysteine lyase) is an enzyme weighing approximately 43.1 kDa. JJU catalyzes the beta replacement reaction of L-cysteine and sulfite to form L-cysteate and hydrogen sulfide<a href="https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10" alt="" target="_blank">[1]</a>.


Usage

The three taurine production pathways incorporated into our E. coli include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway<a href="https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10" alt="" target="_blank">[1]</a>.


Fig. 1. Taurine pathways in E. coli <a href="https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10" alt="" target="_blank">[1]</a>.

JJU is an enzyme that is part of the JJU-CoaBC taurine production pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cysteine to L-cystate, which then becomes taurine by CoaBC<a href="https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10" alt="" target="_blank">[1]</a>.


Fig. 2. Biobrick of Ptrc-jju

JJU enzyme was used in in vitro test of taurine production. The sequence for JJU enzyme and trc promoter (BBa_K864400) were ligated and transformed into E. coli. By adding L-cysteine substrate into a reaction solution, JJU can help catalyze the conversion of L-cysteine to taurine. Taurine concentration were determined by high-performance liquid chromatography (HPLC).


Characterization


The jju sequence synthesized by IDT was amplified by PCR and ligated to trc promoter (BBa_K864400) in the pET28a cloning vector.

Fig. 3. Agarose gel electrophoresis result showing amplified jju gene fragment. M: Marker; Lane 1: jju (1180 bp)



Taurine Production of JJU in E. coli BL21(DE3) strain


In our in vitro test of taurine production by JJU, JJU was transformed into E. coli BL21(DE3) strain. Soluable protein (S) and whole cell (WC) samples were collected to confirm extracellular and intracellular protein expression by SDS-PAGE.

Fig. 4. Confirmation of JJU expression by SDS-PAGE. M: Marker; Lane 1: whole cell of JJU in BL21(DE3); Lane 2: soluble protein of JJU in BL21(DE3) (~43 kDa); Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa)


Whole cell and soluable protein JJU and CoaBC samples were collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. High-performance liquid chromatography (HPLC) was conducted to determine taurine concentration. Because JJU concentration was lower, two times as much JJU soluable protein volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine. As shown in figure 5, 2:1 ratio of JJU to CoaBC soulable protein volume had the highest taurine concentration of around 95 mg/L.

Fig. 5. Taurine production of both JJU and CoaBC in BL21(DE3) in whole cell samples and soluble protein



Taurine Production of JJU in E. coli BD7G strain


Since JJU expression in BL21(DE3) strain was not prominent in the soluable protein, we performed another in vitro test in which PT7-jju was transformed into BD7G strain instead of the BL21(DE3) strain. The BD7G strain contains chaperone protein GroELS that aids in protein folding<a href="https://researchoutput.ncku.edu.tw/zh/publications/enhanced-5-aminolevulinic-acid-production-by-co-expression-of-cod" alt="" target="_blank">[2]</a>. SDS-PAGE results confirm JJU expression in both soluable protein and whole cell samples.

Fig. 6. Confirmation of JJU expression by SDS-PAGE. M: Marker; Lane 1: whole cell of JJU in BD7G; Lane 2: soluble protein of JJU in BD7G (~43 kDa); Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa)


The whole cell and soluable protein JJU and CoaBC samples are collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. When PT7-jju was transformed into the BD7G strain, 1:1 ratio of JJU to CoaBC supernatant had the highest taurine production, as shown in Fig. 7. This suggests the activity level of JJU does not significantly differ from that of CoaBC, and both are equally crucial and effective in converting L-cysteine to taurine.

Fig. 7. Taurine production of JJU in BD7G and CoaBC in BL21(DE3) in soluble protein samples.


References
1.BRENDA - Information on EC 4.4.1.10 - cysteine lyase. Brenda-enzymes.org. Published 2021. Accessed October 6, 2021.
https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10

2.Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli
https://researchoutput.ncku.edu.tw/zh/publications/enhanced-5-aminolevulinic-acid-production-by-co-expression-of-cod

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]