Team:Tsinghua/BioBricks

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<a href="https://2013.igem.org/Team:Tsinghua/BioBricks">BioBricks</a>
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<h1>Biobricks</h1>
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<div id="brief">
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<p>
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Six BioBricks were constructed to engineer a pathogen-detecting yeast strain and benchmark its efficiency. <b>BBa_K1024000</b> and <b>BBa_K1024001</b> were prokaryotic LuxR and pLux BioBricks Parts optimized for yeast expression. <b>BBa_K1024002</b> is a reporter for the LuxR quorum sensing system in yeast, designed to allow engineered yeast to display mCherry fluorescence upon detection of AHL signals. We used this part to test the efficiency of AHL detection. <b>BBa_K1024003</b> is a reporter for the Tet system. <b>BBa_K1024004</b> and <b>BBa_K1024005</b> function as AHL sensor and downstream reportor, respcetively. A protable pathogen detector yeast strain is acquired by mating an a-strain carrying <b>BBa_K1024004</b> and an alpha-strain carrying <b>BBa_K1024005</b>.
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</p>
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</div>
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<div id="section-nav">
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<ul>
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<li class="section1">
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BBa_K1024000
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</li>
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<li class="section2">
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BBa_K1024001
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</li>
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<li class="section3">
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BBa_K1024002
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</li>
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<li class="section4">
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BBa_K1024003
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</li>
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<li class="section5">
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BBa_K1024004
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</li>
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<li class="section6">
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BBa_K1024005
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</li>
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</ul>
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</div>
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<div class="section section1">
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<h2>BBa_K1024000</h2>
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<div class="right" style="width: 400px">
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<img height="auto" src="https://static.igem.org/mediawiki/2013/c/c7/Tsinghua_BBa_K1024000.png" width="100%"/>
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</div>
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<p>
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<b>Part Name:</b> BBa_K1024000
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</p>
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<p>
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<b>Short Description:</b> LuxR in Yeast (pTEF+VP16+NLS+LuxR)
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</p>
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<p>
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<b>Part Type:</b> Regulatory
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</p>
 +
<p>
 +
<b>Design:</b> We constructed and improved standard BioBrick Parts about quorum sensing systems and modified the systems in prokaryotic microorganisms for usage in <i>S. cerevisiae</i> (Yeast). We modified the transcription activator, LuxR (BBa_C0062), by adding nuclear localization signal (NLS) sequence and Herpes simplex virus VP16 activation domain in N-terminus of LuxR, and ligate the sequence of this modified LuxR downstream TEF promoter, which is the constitutive promoter in yeast (BBa_K1024000).
 +
</p>
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<p>
 +
<b>Source:</b> <i>S. cerevisiae</i> & Registry
 +
</p>
 +
<p>
 +
<b>Reference:</b>
 +
<br>[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
 +
<br>[2]Sadowski I, Ma J, Triezenberg S, et al. GAL4-VP16 is an unusually potent transcriptional activator[J]. Nature, 1988, 335(6190): 563-564.
 +
 
 +
</p>
 +
</div>
 +
<div class="section section2">
 +
<h2>BBa_K1024001</h2>
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<div class="right" style="width: 400px">
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<img height="auto" src="https://static.igem.org/mediawiki/2013/6/67/Tsinghua_BBa_K1024001.png" width="100%"/>
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 +
</div>
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<p>
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<b>Part Name:</b> BBa_K1024001
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</p>
 +
<p>
 +
<b>Short Description:</b> Lux Promoter in Yeast (pLux+Cyc Promoter+mCherry)
 +
</p>
 +
<p>
 +
<b>Part Type:</b> Reporter
 +
</p>
 +
<p>
 +
<b>Design:</b> We constructed and improved standard BioBrick Parts about quorum sensing systems and modified the systems in prokaryotic microorganisms for usage in <i>S. cerevisiae</i> (Yeast). We modified the transcriptional regulated promoter in quorum sensing system, Plux promoter (BBa_R0062), by adding cyc100 mini promoter downstream of the Plux promoter (BBa_K1024001).
 +
</p>
 +
<p>
 +
<b>Source:</b> <i>S. cerevisiae</i> & Registry
 +
</p>
 +
<p>
 +
<b>Reference:</b>
 +
<br>[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
 +
<br>[2]Sadowski I, Ma J, Triezenberg S, et al. GAL4-VP16 is an unusually potent transcriptional activator[J]. Nature, 1988, 335(6190): 563-564.
 +
</p>
 +
</div>
 +
<div class="section section3">
 +
<h2>BBa_K1024002</h2>
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<div class="right" style="width: 400px">
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<img height="auto" src="https://static.igem.org/mediawiki/2013/9/9b/Tsinghua_BBa_K1024002.PNG" width="100%"/>
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 +
</div>
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<p>
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<b>Part Name:</b> BBa_K1024002
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</p>
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<p>
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<b>Short Description:</b> Reporter for quorum sensing systems in yeast
 +
</p>
 +
<p>
 +
<b>Part Type:</b> Signaling
 +
</p>
 +
<p>
 +
<b>Design:</b> We constructed and improved standard BioBrick Parts about quorum sensing systems and modified the systems in prokaryotic microorganisms for usage in <i>S. cerevisiae</i> (Yeast). The LuxR gene is constitutively expressed, while the activation of Lux Promoter requires the signaling of N-Acyl Homoserine Lactone (AHL). Therefore, mCherry is activated in the presence of AHL.
 +
</p>
 +
 
 +
<p>
 +
<b>Test:</b> Yeasts after 8 hours of enlarge cultivation were divided into two three groups, induced by DMSO (control), 0.5uM AHL and 200uM AHL separately. After 0.5, 16 and 24 hours, yeasts were collected and tested for mCherry fluorescence by flow cytometry. Total fluorescence were obtained for each group. As shown in the figure, inducing with high concentration of 200uM AHL caused significant increase of mCherry fluorescence. Low concentration of AHL also induced higher fluorescence. Among the three tested time points, the fold change of fluorescence reached the highest when 16 hours after induction.
 +
 +
</p>
 +
<div class="figure">
 +
<img class="center" style="width:400px;height:auto;" src="https://static.igem.org/mediawiki/2013/d/d0/Tsinghua_Flow_Data_Final.jpg"/>
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<p class="legend">
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Figure 1. Fold change of mCherry fluorescence induced by AHL
 +
</p>
 +
 
 +
</div>
 +
<p>
 +
<b>Source:</b> <i>S. cerevisiae</i> & Registry
 +
</p>
 +
<p>
 +
<b>Reference:</b>
 +
<br>[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
 +
<br>[2]Sadowski I, Ma J, Triezenberg S, et al. GAL4-VP16 is an unusually potent transcriptional activator[J]. Nature, 1988, 335(6190): 563-564.
 +
 
 +
</p>
 +
</div>
 +
<div class="section section4">
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<h2>BBa_K1024003</h2>
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<div class="right" style="width: 400px">
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<img height="auto" src="https://static.igem.org/mediawiki/2013/0/07/Tsinghua_BBa_K1024003.PNG" width="100%"/>
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 +
</div>
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<p>
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<b>Part Name:</b> BBa_K1024003
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</p>
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<p>
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<b>Short Description:</b> Reporter for Tet system in Yeast
 +
</p>
 +
<p>
 +
<b>Part Type:</b> Signaling
 +
</p>
 +
<p>
 +
<b>Design:</b> The part is designed to valid the Tet system in yeast. TetR gene with VP16 is constitutively expressed, activating the downstream reporter gene of TetO and CYC1 TATA region. In the yeast (<i>S. cerevisiae</i>) the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment. Thus, it could be used as a marker for screening of target phenotype.
 +
 +
</p>
 +
 
 +
<p>
 +
<b>Test:</b> Yeast with ADE2 knockout exhibits red in color. The function of the plasmid was tested by rescuing the knockout strains, which regained the white color by expressing ADE2.
 +
<div class="figure">
 +
<img class="center" style="width:400px;height:auto;" src="https://static.igem.org/mediawiki/2013/5/54/Tsinghua-part-003.jpg"/>
 +
<p class="legend">
 +
Figure 1. Rescuing the ADE2 knockout yeasts
 +
</p>
 +
 
 +
</div>
 +
</p>
 +
<p>
 +
<b>Source:</b> <i>S. cerevisiae</i> & Registry
 +
</p>
 +
<p>
 +
<b>Reference:</b>
 +
Bellí G, Garí E, Piedrafita L, et al. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast[J]. Nucleic acids research, 1998, 26(4): 942-947.
 +
 
 +
</p>
 +
</div>
 +
<div class="section section5">
 +
<h2>BBa_K1024004</h2>
 +
<div class="right" style="width: 400px">
 +
<img height="auto" src="https://static.igem.org/mediawiki/2013/0/00/Tsinghua_BBa_K1024004.PNG" width="100%"/>
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</div>
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<p>
 +
<b>Part Name:</b> BBa_K1024004
 +
</p>
 +
<p>
 +
<b>Short Description:</b> Sensor yeast (inducible TetR+VP16)
 +
</p>
 +
<p>
 +
<b>Part Type:</b> Signaling
 +
</p>
 +
<p>
 +
<b>Design:</b> The part was the sensor of the pathogen detector. LuxR was constitutively expressed. When sensing AHL, LuxR will bind to the Lux promoter and thus activated the downstream TetR with VP16. Mated with reporter yeast (BBa_K1024005), the TetR with VP16 will bind to the Tet operator and activated the downstream gene.
 +
</p>
 +
 
 +
 
 +
 
 +
<p>
 +
<b>Source:</b> <i>S. cerevisiae</i> & Registry
 +
</p>
 +
<p>
 +
<b>Reference:</b>
 +
<br>[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
 +
<br>[2]G, Garí E, Piedrafita L, et al. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast[J]. Nucleic acids research, 1998, 26(4): 942-947.
 +
 
 +
</p>
 +
</div>
 +
<div class="section section6">
 +
<h2>BBa_K1024005</h2>
 +
<div class="right" style="width: 400px">
 +
<img height="auto" src="https://static.igem.org/mediawiki/2013/3/3d/Tsinghua_BBa_K1024005.PNG" width="100%"/>
 +
</div>
 +
<p>
 +
<b>Part Name:</b> BBa_K1024005
 +
</p>
 +
<p>
 +
<b>Short Description:</b> Report yeast (inducible ADE2)
 +
</p>
 +
<p>
 +
<b>Part Type:</b> Signaling
 +
</p>
 +
<p>
 +
<b>Design:</b> The part was the reporter of the pathogen detector. Tet operator and CYC1 TATA region was followed by ADE2 gene. In the yeast <i>S. cerevisiae</i> the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment. Mated with sensor yeast (BBa_K1024004) which is induced by AHL, the ADE2 gene will be activated and rescuing the ADE2 knockout yeast by making it white.
 +
</p>
 +
 
 +
 
 +
<p>
 +
<b>Source:</b> <i>S. cerevisiae</i> & Registry
 +
</p>
 +
<p>
 +
<b>Reference:</b>
 +
<br>[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
 +
<br>[2]Bellí G, Garí E, Piedrafita L, et al. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast[J]. Nucleic acids research, 1998, 26(4): 942-947.
 +
</p>
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</div>
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<script type="text/javascript">
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Latest revision as of 21:00, 27 September 2013

Biobricks

Six BioBricks were constructed to engineer a pathogen-detecting yeast strain and benchmark its efficiency. BBa_K1024000 and BBa_K1024001 were prokaryotic LuxR and pLux BioBricks Parts optimized for yeast expression. BBa_K1024002 is a reporter for the LuxR quorum sensing system in yeast, designed to allow engineered yeast to display mCherry fluorescence upon detection of AHL signals. We used this part to test the efficiency of AHL detection. BBa_K1024003 is a reporter for the Tet system. BBa_K1024004 and BBa_K1024005 function as AHL sensor and downstream reportor, respcetively. A protable pathogen detector yeast strain is acquired by mating an a-strain carrying BBa_K1024004 and an alpha-strain carrying BBa_K1024005.

  • BBa_K1024000
  • BBa_K1024001
  • BBa_K1024002
  • BBa_K1024003
  • BBa_K1024004
  • BBa_K1024005

BBa_K1024000

Part Name: BBa_K1024000

Short Description: LuxR in Yeast (pTEF+VP16+NLS+LuxR)

Part Type: Regulatory

Design: We constructed and improved standard BioBrick Parts about quorum sensing systems and modified the systems in prokaryotic microorganisms for usage in S. cerevisiae (Yeast). We modified the transcription activator, LuxR (BBa_C0062), by adding nuclear localization signal (NLS) sequence and Herpes simplex virus VP16 activation domain in N-terminus of LuxR, and ligate the sequence of this modified LuxR downstream TEF promoter, which is the constitutive promoter in yeast (BBa_K1024000).

Source: S. cerevisiae & Registry

Reference:
[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
[2]Sadowski I, Ma J, Triezenberg S, et al. GAL4-VP16 is an unusually potent transcriptional activator[J]. Nature, 1988, 335(6190): 563-564.

BBa_K1024001

Part Name: BBa_K1024001

Short Description: Lux Promoter in Yeast (pLux+Cyc Promoter+mCherry)

Part Type: Reporter

Design: We constructed and improved standard BioBrick Parts about quorum sensing systems and modified the systems in prokaryotic microorganisms for usage in S. cerevisiae (Yeast). We modified the transcriptional regulated promoter in quorum sensing system, Plux promoter (BBa_R0062), by adding cyc100 mini promoter downstream of the Plux promoter (BBa_K1024001).

Source: S. cerevisiae & Registry

Reference:
[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
[2]Sadowski I, Ma J, Triezenberg S, et al. GAL4-VP16 is an unusually potent transcriptional activator[J]. Nature, 1988, 335(6190): 563-564.

BBa_K1024002

Part Name: BBa_K1024002

Short Description: Reporter for quorum sensing systems in yeast

Part Type: Signaling

Design: We constructed and improved standard BioBrick Parts about quorum sensing systems and modified the systems in prokaryotic microorganisms for usage in S. cerevisiae (Yeast). The LuxR gene is constitutively expressed, while the activation of Lux Promoter requires the signaling of N-Acyl Homoserine Lactone (AHL). Therefore, mCherry is activated in the presence of AHL.

Test: Yeasts after 8 hours of enlarge cultivation were divided into two three groups, induced by DMSO (control), 0.5uM AHL and 200uM AHL separately. After 0.5, 16 and 24 hours, yeasts were collected and tested for mCherry fluorescence by flow cytometry. Total fluorescence were obtained for each group. As shown in the figure, inducing with high concentration of 200uM AHL caused significant increase of mCherry fluorescence. Low concentration of AHL also induced higher fluorescence. Among the three tested time points, the fold change of fluorescence reached the highest when 16 hours after induction.

Figure 1. Fold change of mCherry fluorescence induced by AHL

Source: S. cerevisiae & Registry

Reference:
[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
[2]Sadowski I, Ma J, Triezenberg S, et al. GAL4-VP16 is an unusually potent transcriptional activator[J]. Nature, 1988, 335(6190): 563-564.

BBa_K1024003

Part Name: BBa_K1024003

Short Description: Reporter for Tet system in Yeast

Part Type: Signaling

Design: The part is designed to valid the Tet system in yeast. TetR gene with VP16 is constitutively expressed, activating the downstream reporter gene of TetO and CYC1 TATA region. In the yeast (S. cerevisiae) the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment. Thus, it could be used as a marker for screening of target phenotype.

Test: Yeast with ADE2 knockout exhibits red in color. The function of the plasmid was tested by rescuing the knockout strains, which regained the white color by expressing ADE2.

Figure 1. Rescuing the ADE2 knockout yeasts

Source: S. cerevisiae & Registry

Reference: Bellí G, Garí E, Piedrafita L, et al. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast[J]. Nucleic acids research, 1998, 26(4): 942-947.

BBa_K1024004

Part Name: BBa_K1024004

Short Description: Sensor yeast (inducible TetR+VP16)

Part Type: Signaling

Design: The part was the sensor of the pathogen detector. LuxR was constitutively expressed. When sensing AHL, LuxR will bind to the Lux promoter and thus activated the downstream TetR with VP16. Mated with reporter yeast (BBa_K1024005), the TetR with VP16 will bind to the Tet operator and activated the downstream gene.

Source: S. cerevisiae & Registry

Reference:
[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
[2]G, Garí E, Piedrafita L, et al. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast[J]. Nucleic acids research, 1998, 26(4): 942-947.

BBa_K1024005

Part Name: BBa_K1024005

Short Description: Report yeast (inducible ADE2)

Part Type: Signaling

Design: The part was the reporter of the pathogen detector. Tet operator and CYC1 TATA region was followed by ADE2 gene. In the yeast S. cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment. Mated with sensor yeast (BBa_K1024004) which is induced by AHL, the ADE2 gene will be activated and rescuing the ADE2 knockout yeast by making it white.

Source: S. cerevisiae & Registry

Reference:
[1]Fuqua W C, Winans S C, Greenberg E P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[J]. Journal of bacteriology, 1994, 176(2): 269.
[2]Bellí G, Garí E, Piedrafita L, et al. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast[J]. Nucleic acids research, 1998, 26(4): 942-947.

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