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

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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.  
 
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 system⁴ 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 is equipped with a promoter from E. coli, which endogenously regulates transcription of the acid shock RNA (asr) gene through the phoB-phoR regulatory system⁴ 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]]
  
 
For our pTurquoise plasmid we are taking advantage of an E. coli sodium and proton anti-transport promoter,¹ 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, mTurquoise2³ (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.
 
For our pTurquoise plasmid we are taking advantage of an E. coli sodium and proton anti-transport promoter,¹ 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, mTurquoise2³ (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===

Revision as of 21:08, 10 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 system⁴ 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,¹ 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, mTurquoise2³ (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).

References

1. Yang, Chih-Hsien. "Part:BBa_K118021." Part:BBa K118021 - Parts.igem.org. IGEM, 29 Oct. 2008. Web. 10 May 2017.

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

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

4. Team:NYMU-Taipei/Project. N.p., n.d. Web. 10 June 2017.