Difference between revisions of "Part:BBa K3771000"
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<br><b style="font-size:1.3rem">Biology</b> | <br><b style="font-size:1.3rem">Biology</b> | ||
| − | <p>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[1,2].</p> | + | <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> |
<div style="width=100%; display:flex; align-items: center; justify-content: center;"> | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> | ||
<img src="圖片網址" style="width:35%;"> | <img src="圖片網址" style="width:35%;"> | ||
</div> | </div> | ||
| − | <p align="center">Figure.1 Taurine pathways in E. coli [1,2].</p> | + | <p align="center">Figure.1 Taurine pathways in <i>E. coli<i> [1,2].</p> |
<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> | <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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<img src="圖片網址" style="width:35%;"> | <img src="圖片網址" style="width:35%;"> | ||
</div> | </div> | ||
| − | <p align="center">Figure 2. Biobrick of trc-JJU</p> | + | <p align="center">Figure 2. Biobrick of <i>trc<i>-JJU</p> |
| − | <p>JJU enzyme was used in in vitro testing 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 concentrations were determined using high-performance liquid chromatography (HPLC).</p> | + | <p>JJU enzyme was used in <i>in vitro<i> testing of taurine production. The sequence for JJU enzyme and <i>trc<i> promoter (BBa_K864400) 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> |
<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">Figure 3. Agarose gel electrophoresis results showing amplified jju gene fragment</p> | <p align="center">Figure 3. Agarose gel electrophoresis results showing amplified jju gene fragment</p> | ||
| − | <br><b style="font-size:1.1rem">Taurine Production of JJU in E. coli BL21(DE3) strain</b> | + | <br><b style="font-size:1.1rem">Taurine Production of JJU in <i>E. coli<i> BL21(DE3) strain</b> |
<br> | <br> | ||
| − | <br>In our in vitro testing of taurine production by JJU, JJU was transformed into E. coli BL21(DE3) strain. Supernatant (S) and whole cell (WC) samples were collected to confirm extracellular and intracellular protein expression by SDS-PAGE. <br> | + | <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> |
<div style="width=100%; display:flex; align-items: center; justify-content: center;"> | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> | ||
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<img src="圖片網址" style="width:35%;"> | <img src="圖片網址" style="width:35%;"> | ||
</div> | </div> | ||
| − | <p align="center">Figure 5. (in vitro 1 taurine)</p> | + | <p align="center">Figure 5. (<i>in vitro<i> 1 taurine)</p> |
<br><b style="font-size:1.1rem">Taurine Production of JJU in E. coli BD7G strain</b> | <br><b style="font-size:1.1rem">Taurine Production of JJU in E. coli BD7G strain</b> | ||
<br> | <br> | ||
| − | <br>Since JJU expression in BL21(DE3) strain was not prominent in the supernatant, 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 [1]. SDS-PAGE results confirm JJU expression in both supernatant and whole cell samples.<br> | + | <br>Since JJU expression in BL21(DE3) strain was not prominent in the supernatant, we performed another <i>in vitro<i> 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 [1]. SDS-PAGE results confirm JJU expression in both supernatant and whole cell samples.<br> |
<div style="width=100%; display:flex; align-items: center; justify-content: center;"> | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> | ||
<img src="圖片網址" style="width:35%;"> | <img src="圖片網址" style="width:35%;"> | ||
</div> | </div> | ||
| − | <p align="center">Figure 6. (PAGE in vitro 2)</p> | + | <p align="center">Figure 6. (PAGE <i>in vitro<i> 2)</p> |
<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 PT7-jju 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> | <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 PT7-jju 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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<img src="圖片網址" style="width:35%;"> | <img src="圖片網址" style="width:35%;"> | ||
</div> | </div> | ||
| − | <p align="center">Figure 7. (in vitro 2 taurine)</p> | + | <p align="center">Figure 7. (<i>in vitro<i> 2 taurine)</p> |
<br><b style="font-size:1.3rem">References</b> | <br><b style="font-size:1.3rem">References</b> | ||
Revision as of 05:43, 17 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[1].
Biology
The three taurine production pathways incorporated into our E. coli<i> include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway[1,2].</p>
<img src="圖片網址" style="width:35%;">
<p align="center">Figure.1 Taurine pathways in <i>E. coli<i> [1,2].</p>
<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>
Usage
<img src="圖片網址" style="width:35%;">
<p align="center">Figure 2. Biobrick of <i>trc<i>-JJU</p> <p>JJU enzyme was used in <i>in vitro<i> testing of taurine production. The sequence for JJU enzyme and <i>trc<i> promoter (BBa_K864400) 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>
Characterization
<p>
The jju sequence synthesized by IDT was amplified by PCR and ligated to trc promoter (BBa_K864400) in the pET28a cloning vector.
<img src="圖片網址" style="width:35%;">
<p align="center">Figure 3. Agarose gel electrophoresis results showing amplified jju gene fragment</p>
Taurine Production of JJU in <i>E. coli<i> BL21(DE3) strain
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.
<img src="圖片網址" style="width:35%;">
<p align="center">Figure 4. Confirmation of JJU expression by SDS-PAGE</p>
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.
<img src="圖片網址" style="width:35%;">
<p align="center">Figure 5. (<i>in vitro<i> 1 taurine)</p>
Taurine Production of JJU in E. coli BD7G strain
Since JJU expression in BL21(DE3) strain was not prominent in the supernatant, we performed another <i>in vitro<i> 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 [1]. SDS-PAGE results confirm JJU expression in both supernatant and whole cell samples.
<img src="圖片網址" style="width:35%;">
<p align="center">Figure 6. (PAGE <i>in vitro<i> 2)</p>
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 PT7-jju 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.
<img src="圖片網址" style="width:35%;">
<p align="center">Figure 7. (<i>in vitro<i> 2 taurine)</p>
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. Sung Ok Han et. al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. J. Agric. Food Chem. 2018, 66, 13454−13463
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]