Difference between revisions of "Part:BBa K3771075"
 
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<br>Trc promoter constitutively facilitates the expression of CDO1 enzyme.
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  <br><i>trc</i> promoter constitutively facilitates the expression of CDO1 enzyme.
 
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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<img src="https://2021.igem.org/wiki/images/4/4c/T--NCKU_Tainan--taurine_pathway.png" style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/4/4c/T--NCKU_Tainan--taurine_pathway.png" style="width:50%;">
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</div><p align="center">Fig. 1. Taurine production pathway</p>
  
  
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<br><b style="font-size:1.3rem">Usage</b>
 
<br><b style="font-size:1.3rem">Usage</b>
 
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<br>CDO1 was used in in vivo testing of taurine production. The sequence for CDO1 enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC). <br>
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  <br>CDO1 was used in <i>in vivo</i> testing of taurine production. The sequence for CDO1 enzyme and <i>trc</i> promoter were ligated and transformed into <i>E. coli</i> to calculate taurine production using high-performance liquid chromatography (HPLC). <br>
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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<img src="https://2021.igem.org/wiki/images/a/aa/T--NCKU_Tainan--3.png" style="width:35%;">
 
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<p>Fig. 2 Taurine production of Ptrc-cdo1 +PlacI-csad +Ptrc-cs in different growth mediums.</p>
 
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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<img src="https://2021.igem.org/wiki/images/6/6b/T--NCKU_Tainan--invivo1.png" style="width:35%;">
 
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<p>Fig. 3 Taurine production of Ptrc-cdo1+Ptrc-csad in different growth mediums.</p>
 
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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<img src="https://2021.igem.org/wiki/images/f/f1/T--NCKU_Tainan--DkO.png" style="width:35%;">
 
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<p>Fig. 4 Taurine production (extracellular) of CDO1 and CSAD in <i>∆tauD</i>.</p>
 
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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<img src="https://2021.igem.org/File:T--NCKU_Tainan--tauD2.png" style="width:35%;">
 
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<p>Fig. 5 Taurine production (extracellular) of CDO1 and CSAD in DkO.</p>
 
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<img src="https://2021.igem.org/wiki/images/d/dd/T--NCKU_Tainan--in.png" style="width:35%;">
 
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<p>Fig. 6 Taurine production (intracellular and extracellular) of CDO1 and CSAD in DkO and top10.</p>
 
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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<img src="https://2021.igem.org/wiki/images/2/22/T--NCKU_Tainan--invivo4.png" style="width:35%;">
 
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<p>Fig. 7  Taurine production (extracellular) of CDO1 and CSAD with expression of CSAD regulated by soxS promoter in DkO.</p>
 
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<img src="https://2021.igem.org/wiki/images/a/ab/T--NCKU_Tainan--CDO1-PCR.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/a/ab/T--NCKU_Tainan--CDO1-PCR.png" style="width:35%;">
 
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<p align="center">Fig. 8 Confirmation of cdo1 fragment by PCR. M: Marker; Lane 1: cdo1 (603 bp)</p>
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  <p align="center">Fig. 2. Confirmation of <i>cdo1</i> fragment by PCR. M: Marker; Lane 1: <i>cdo1</i> (603 bp)</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="https://2021.igem.org/wiki/images/9/9d/T--NCKU_Tainan--CDO1-digestion.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/9/9d/T--NCKU_Tainan--CDO1-digestion.png" style="width:35%;">
 
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<p align="center">Fig. 9 Confirmation of  pSB4KI-Ptrc-CDO1 by digestion.
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  <p align="center">Fig. 3. Confirmation of  pSB4KI-<i>P<sub>trc</sub>-cdo1</i> by digestion.
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M: Marker; Lane 1~3: Different colonies of pSB4KI-Ptrc-CDO1 (5470 bp)</p>
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    M: Marker; Lane 1~3: Different colonies of pSB4KI-<i>P<sub>trc</sub>-cdo1</i> (5470 bp)</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="https://2021.igem.org/wiki/images/9/90/T--NCKU_Tainan--CDO1-plate%28DH5a%29.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/9/90/T--NCKU_Tainan--CDO1-plate%28DH5a%29.png" style="width:35%;">
 
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<p align="center">Fig. 10 Transformation / CDO1 in DH5α</p>
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<p align="center">Fig. 4. Transformation / CDO1 in DH5α</p>
  
 
<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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<img src="https://2021.igem.org/wiki/images/4/4e/T--NCKU_Tainan--CDO1-PAGE.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/4/4e/T--NCKU_Tainan--CDO1-PAGE.png" style="width:35%;">
 
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<p align="center">Fig. 11 Confirmation of protein expression of CDO1.
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<p align="center">Fig. 5. Confirmation of protein expression of CDO1.
 
M: Marker; Lane1: CDO1 in DH5α without induction; Lane2: CDO1 in DH5α with induction (~22 kDa)</p>
 
M: Marker; Lane1: CDO1 in DH5α without induction; Lane2: CDO1 in DH5α with induction (~22 kDa)</p>
 
    
 
    
 
    
 
    
 
    
 
    
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<br>In Vivo Test 1: Choosing a Growth Medium
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  <br><i>In Vivo</i> Test 1: Choosing a Growth Medium
 
Since the intestine is not as nutrient-rich as LB (lysogeny broth), we chose to test taurine production in both LB and M9 (minimal salts) mediums. M9 consists of fewer nutrients than LB and more closely mimics the nutrient composition of the human intestine.
 
Since the intestine is not as nutrient-rich as LB (lysogeny broth), we chose to test taurine production in both LB and M9 (minimal salts) mediums. M9 consists of fewer nutrients than LB and more closely mimics the nutrient composition of the human intestine.
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First, Ptrc-cdo1 (BBa_K3771075), PlacI-csad (BBa_K3771076), and Ptrc-cs (BBa_K3771078) were transformed into DH5α strain. In another sample, only Ptrc-cdo1 and Ptrc-csad (BBa_K3771077) were transformed. The two samples were each grown in LB with IPTG inducer (LB+), M9 with IPTG inducer (M9+) , and M9 without IPTG inducer (M9-) mediums.  
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  First, <i>P<sub>trc</sub>-cdo1</i> (BBa_K3771075), <i>P<sub>lacI</sub>-csad</i> (BBa_K3771076), and <i>P<sub>trc</sub>-cs</i> (BBa_K3771078) were transformed into DH5α strain. In another sample, only <i>P<sub>trc</sub>-cdo1</i> and <i>P<sub>trc</sub>-csad</i> (BBa_K3771077) were transformed. The two samples were each grown in LB with IPTG inducer (LB+), M9 with IPTG inducer (M9+) , and M9 without IPTG inducer (M9-) mediums.  
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The samples’ respective growth curves are shown in figure 21 and 22. Interestingly, the sample with all three enzymes (Ptrc-cdo1 +PlacI-csad +Ptrc-cs) had a lower growth rate in M9+ medium than in M9- medium. In the sample with two enzymes (Ptrc-cdo1+Ptrc-csad), M9 medium with or without an inducer added produced similar growth curves. Both samples grew best in LB (Fig. 11, 12)<br>
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The samples’ respective growth curves are shown in figure 12 and 13. Interestingly, the sample with all three enzymes (Ptrc-cdo1 +PlacI-csad +Ptrc-cs) had a lower growth rate in M9+ medium than in M9- medium. In the sample with two enzymes (Ptrc-cdo1+Ptrc-csad), M9 medium with or without an inducer added produced similar growth curves. Both samples grew best in LB (Fig. 12, 13)<br>
 
    
 
    
 
    
 
    
 
      
 
      
 
  <div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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  <img src="https://2021.igem.org/wiki/images/4/4e/T--NCKU_Tainan--3growth.png" style="width:35%;">
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  <img src="https://2021.igem.org/wiki/images/b/b6/T--NCKU_Tainan--4.png" style="width:35%;">
 
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<p>Fig. 12 Growth curve of Ptrc-cdo1 +PlacI-csad +Ptrc-cs in different growth mediums.</p>
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  <p align="center">Fig. 6. Growth curve of <i>P<sub>trc</sub>-cdo1</i> +<i>P<sub>lacI</sub>-csad</i> +<i>P<sub>trc</sub>-cs</i> in different growth mediums.</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;">
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  <img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--invivo-growth.png" style="width:35%;">
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  <img src="https://2021.igem.org/wiki/images/6/6e/T--NCKU_Tainan--5-5.png" style="width:35%;">
 
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<p>Fig. 13 Growth curve of Ptrc-cdo1+Ptrc-csad in different growth mediums.</p>
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  <p align="center">Fig. 7. Growth curve of <i>P<sub>trc</sub>-cdo1</i>+<i>P<sub>trc</sub>-csad</i> in different growth mediums.</p>
 
    
 
    
  
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<br><b style="font-size:1.3rem">References</b>
 
<br><b style="font-size:1.3rem">References</b>
 
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  <br>1. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093<br>
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===Usage and Biology===
 
===Usage and Biology===
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3771075 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3771075 SequenceAndFeatures</partinfo>

Latest revision as of 03:50, 22 October 2021


Ptrc-CDO1


Description

Ptrc-cdo1 is a composite part consisting of the trc promoter and the cdo1 sequences. This part was used in in vivo testing of taurine production. The sequence for cdo1 and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Biology

trc promoter constitutively facilitates the expression of CDO1 enzyme.

Fig. 1. Taurine production pathway


Usage

CDO1 was used in in vivo testing of taurine production. The sequence for CDO1 enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Characterization

Fig. 2. Confirmation of cdo1 fragment by PCR. M: Marker; Lane 1: cdo1 (603 bp)

Fig. 3. Confirmation of pSB4KI-Ptrc-cdo1 by digestion. M: Marker; Lane 1~3: Different colonies of pSB4KI-Ptrc-cdo1 (5470 bp)

Fig. 4. Transformation / CDO1 in DH5α

Fig. 5. Confirmation of protein expression of CDO1. M: Marker; Lane1: CDO1 in DH5α without induction; Lane2: CDO1 in DH5α with induction (~22 kDa)


In Vivo Test 1: Choosing a Growth Medium Since the intestine is not as nutrient-rich as LB (lysogeny broth), we chose to test taurine production in both LB and M9 (minimal salts) mediums. M9 consists of fewer nutrients than LB and more closely mimics the nutrient composition of the human intestine. First, Ptrc-cdo1 (BBa_K3771075), PlacI-csad (BBa_K3771076), and Ptrc-cs (BBa_K3771078) were transformed into DH5α strain. In another sample, only Ptrc-cdo1 and Ptrc-csad (BBa_K3771077) were transformed. The two samples were each grown in LB with IPTG inducer (LB+), M9 with IPTG inducer (M9+) , and M9 without IPTG inducer (M9-) mediums. The samples’ respective growth curves are shown in figure 12 and 13. Interestingly, the sample with all three enzymes (Ptrc-cdo1 +PlacI-csad +Ptrc-cs) had a lower growth rate in M9+ medium than in M9- medium. In the sample with two enzymes (Ptrc-cdo1+Ptrc-csad), M9 medium with or without an inducer added produced similar growth curves. Both samples grew best in LB (Fig. 12, 13)

Fig. 6. Growth curve of Ptrc-cdo1 +PlacI-csad +Ptrc-cs in different growth mediums.

Fig. 7. Growth curve of Ptrc-cdo1+Ptrc-csad in different growth mediums.


References

1. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
Sequence and Features


Assembly Compatibility:
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    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 534
  • 1000
    COMPATIBLE WITH RFC[1000]