Difference between revisions of "Part:BBa M50073:Design"

 
 
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===Design Notes===
 
===Design Notes===
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We engineered pPink to have an ampicillin selection marker with a low copy number of origin of replication (PJ-Amp_Low). We engineered pTurquoise to have a kanamycin resistance with a high copy number of origin of replication (pJ-Kan_High). Since we thought the turquoise fluorescent protein might be more difficult to visualize, we chose to upregulate its production as we could not have a high copy number for both plasmids if inserted in the same organism.  
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In order to sense both fairly acidic and fairly basic pHs that could be a product of ocean acidification, we have designed two plasmids, pPink and pTurquoise, that produce their respective fluorescent colored proteins in response to two pH ranges.
 +
pPink is equipped with a promoter from E. coli, which endogenously regulates transcription of the acid shock RNA (asr) gene through the phoB-phoR regulatory system¹ and is inducible by pHs ranging from 4.8 to 7 (iGEM Part: BBa_E1010). We will use DNA 2.0’s default strong RBS, in order to maximize the production of our pink fluorescent protein, FresnoRFP,² which is also a DNA 2.0 product. Attached to the gene for our pink fluorescent protein is a FLAG tag, and a transcription terminator sequence (pA-GH-Bt). We engineered pPink to have an ampicillin selection marker with a low copy number of origin of replication (PJ-Amp_Low).  
  
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[[File:pPink Schematic.png]]
  
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For our pTurquoise plasmid we are taking advantage of an E. coli sodium and proton anti-transport promoter,³ which can be used as an inducible-pH sensor for pH’s ranging from 5.5 to 8 (iGEM Part: BBa_K116001). We also used DNA 2.0’s default strong RBS, in order to maximize the production of our turquoise fluorescent protein, mTurquoise2⁴ (iGEM Part: BBa_M5002). Attached to our turquoise fluorescent protein is a 6xHIS tag, and a transcription terminator sequence (pA-GH-Bt). We engineered pTurquoise to have a kanamycin resistance with a high copy number of origin of replication (pJ-Kan_High). Since we thought the turquoise fluorescent protein might be more difficult to visualize, we chose to upregulate its production as we could not have a high copy number for both plasmids if inserted in the same organism. Reference Figure 2 below for a visual schematic of our pTurquoise design.
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[[File:pTurquoise Schematic.png]]
  
 
===Source===
 
===Source===
  
 
For our promoter in pTurquoise, we used iGEM part BBa_K116001. The turquoise fluorescent protein iGEM part name is BBa_M50029. Following this we used a His tag and a terminator (pA-GH-Bt). For our promoter in pPink, we used iGEM part BBa_K1170000. The RFP iGEM part name is BBa_E1010. Following this we used a FLAG tag and a terminator (pA-GH-Bt).
 
For our promoter in pTurquoise, we used iGEM part BBa_K116001. The turquoise fluorescent protein iGEM part name is BBa_M50029. Following this we used a His tag and a terminator (pA-GH-Bt). For our promoter in pPink, we used iGEM part BBa_K1170000. The RFP iGEM part name is BBa_E1010. Following this we used a FLAG tag and a terminator (pA-GH-Bt).
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See pPink and pTurquoise pages for sequence information.
  
 
===References===
 
===References===
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1. Šeputienė, Vaida, Domantas Motiejūnas, Kęstutis Sužiedėlis, Henrik Tomenius, Staffan Normark, Öjar Melefors, and Edita Sužiedėlienė. "Molecular Characterization of the Acid-Inducible asr Gene of Escherichia Coli and Its Role in Acid Stress Response." Journal of Bacteriology. American Society for Microbiology, Apr. 2003. Web. 10 June 2017.
 +
 +
2. "ProteinPaintbox®." ATUM. ATUM, n.d. Web. 10 May 2017.
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 +
3. Yang, Chih-Hsien. "Part:BBa_K116001." Part:BBa K116001 - Parts.igem.org. IGEM, 29 Oct. 2008. Web. 10 May 2017.
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 +
4.  Mahajan, Vinay S. "Part:BBa_B0034." IGEM, 31 Jan. 2003. Web. 9 May 2017.

Latest revision as of 05:48, 12 June 2017


E. coli pH sensor


Assembly Compatibility:
  • 10
    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 AgeI site found at 1747
    Illegal AgeI site found at 1859
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In order to sense both fairly acidic and fairly basic pHs that could be a product of ocean acidification, we have designed two plasmids, pPink and pTurquoise, that produce their respective fluorescent colored proteins in response to two pH ranges. pPink is equipped with a promoter from E. coli, which endogenously regulates transcription of the acid shock RNA (asr) gene through the phoB-phoR regulatory system¹ and is inducible by pHs ranging from 4.8 to 7 (iGEM Part: BBa_E1010). We will use DNA 2.0’s default strong RBS, in order to maximize the production of our pink fluorescent protein, FresnoRFP,² which is also a DNA 2.0 product. Attached to the gene for our pink fluorescent protein is a FLAG tag, and a transcription terminator sequence (pA-GH-Bt). We engineered pPink to have an ampicillin selection marker with a low copy number of origin of replication (PJ-Amp_Low).

PPink Schematic.png

For our pTurquoise plasmid we are taking advantage of an E. coli sodium and proton anti-transport promoter,³ which can be used as an inducible-pH sensor for pH’s ranging from 5.5 to 8 (iGEM Part: BBa_K116001). We also used DNA 2.0’s default strong RBS, in order to maximize the production of our turquoise fluorescent protein, mTurquoise2⁴ (iGEM Part: BBa_M5002). Attached to our turquoise fluorescent protein is a 6xHIS tag, and a transcription terminator sequence (pA-GH-Bt). We engineered pTurquoise to have a kanamycin resistance with a high copy number of origin of replication (pJ-Kan_High). Since we thought the turquoise fluorescent protein might be more difficult to visualize, we chose to upregulate its production as we could not have a high copy number for both plasmids if inserted in the same organism. Reference Figure 2 below for a visual schematic of our pTurquoise design.

PTurquoise Schematic.png

Source

For our promoter in pTurquoise, we used iGEM part BBa_K116001. The turquoise fluorescent protein iGEM part name is BBa_M50029. Following this we used a His tag and a terminator (pA-GH-Bt). For our promoter in pPink, we used iGEM part BBa_K1170000. The RFP iGEM part name is BBa_E1010. Following this we used a FLAG tag and a terminator (pA-GH-Bt).

See pPink and pTurquoise pages for sequence information.

References

1. Šeputienė, Vaida, Domantas Motiejūnas, Kęstutis Sužiedėlis, Henrik Tomenius, Staffan Normark, Öjar Melefors, and Edita Sužiedėlienė. "Molecular Characterization of the Acid-Inducible asr Gene of Escherichia Coli and Its Role in Acid Stress Response." Journal of Bacteriology. American Society for Microbiology, Apr. 2003. Web. 10 June 2017.

2. "ProteinPaintbox®." ATUM. ATUM, n.d. Web. 10 May 2017.

3. Yang, Chih-Hsien. "Part:BBa_K116001." Part:BBa K116001 - Parts.igem.org. IGEM, 29 Oct. 2008. Web. 10 May 2017.

4. Mahajan, Vinay S. "Part:BBa_B0034." IGEM, 31 Jan. 2003. Web. 9 May 2017.