Difference between revisions of "Part:BBa M36532:Design"

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===Design Notes===
 
===Design Notes===
  
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Below is an example of a possible implementation of BBa_M36532, the Arsenate Bioremediation Actuator. Due to the necessity of including genes PHO84, ACR 1, and ACR 2 and size limitations, it was necessary to design two separate constructs: one with the PHO84 and another with ACR 1 and 2. In this example, a GAL1 inducible sensor has been attached upstream for both designs. This image shows the different components of the Arsenate Bioremediation Actuator. Part numbers for the Kozak sequence and the PHO84, ACR 1, and ACR 2 genes can be found in the following section.  
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Below is an example of a possible implementation of BBa_M36532, the Arsenate Bioremediation Actuator. Due to size limitations, PHO84, ACR 1, and ACR 2, this actuator was created in two constructs,  one with the PHO84 (M36533)[https://parts.igem.org/Part:BBa_M36533] and another with ACR 1 and 2 (M36534) [https://parts.igem.org/Part:BBa_M36534]. In this example, a GAL1 inducible sensor has been attached upstream of both designs. This image shows the different components of the Arsenate Bioremediation Actuator.  
  
  
 
Example implementation:
 
Example implementation:
  
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http://imageshack.com/a/img907/2517/DITZFh.png
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http://i67.tinypic.com/153xj49.png
  
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http://imageshack.com/a/img907/9338/NX12gL.png
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'''Glycerol Freezer Stock'''
 +
 
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Plasmid: ACR2 ACR1
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 +
Barcode: 0133027024
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Unique ID: HTALE
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Plasmid: PHO84
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Barcode: 0133029030
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Unique ID: PKPHO8411/18/15
  
 
===Source===
 
===Source===
  
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DNA 2.0 Plasmid (pD1201) with GAL1 inducible promoter, high copy number, and Uracil as the selectable marker.
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DNA 2.0 plasmid backbone (pD1201) with GAL1 inducible promoter, high copy number ORI, and ampicillin and leucine selectable markers.
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[https://www.dna20.com/eCommerce/catalog/datasheet/188]
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DNA 2.0 Plasmid (pD1204) with GAL1 inducible promoter, high copy number, and Uracil as the selectable marker.
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DNA 2.0 for plasmid backbone (pD1207) with GAL1 inducible promoter, high copy number ORI, and kanamycin and histidine selectable markers.
 
[https://www.dna20.com/eCommerce/catalog/datasheet/189]
 
[https://www.dna20.com/eCommerce/catalog/datasheet/189]
  
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RBS consensus yeast Kozak sequence taken from the Silver Lab, BBa_J63003 (Partsregistry.org), was used for RBS1 and 2 on both constructs.  
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In order to have a multi-protein expression cassette, we incorporated an Internal Ribosome Entry Site (IRES), which was a T2A sequence. We got the amino acid sequence for this part from the following site [http://blog.addgene.org/plasmids-101-multicistronic-vectors]
  
 
Naturally occurring genes in S. cerevisiae: Pho84, ACR 1 and 2
 
Naturally occurring genes in S. cerevisiae: Pho84, ACR 1 and 2
 
Sequences were referenced from the yeast genome website and then were entered into the registry.  
 
Sequences were referenced from the yeast genome website and then were entered into the registry.  
  
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PHO84: BBa_M36535 (Partsregistry.org)
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PHO84: BBa_M36535 [https://parts.igem.org/Part:BBa_M36535]
  
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ACR 1: BBa_M36536 (Partsregistry.org)
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ACR 1: BBa_M36536 [https://parts.igem.org/Part:BBa_M36536]
  
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ACR 2: BBa_M36537 (Partsregistry.org)
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ACR 2: BBa_M36537 [https://parts.igem.org/Part:BBa_M36537]
  
 
===References===
 
===References===

Latest revision as of 10:28, 9 December 2015

Arsenate Bioremediation Actuator in S. Cerevisiae


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2711
    Illegal XbaI site found at 1680
    Illegal PstI site found at 1035
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2711
    Illegal PstI site found at 1035
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2711
    Illegal BglII site found at 1471
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2711
    Illegal XbaI site found at 1680
    Illegal PstI site found at 1035
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2711
    Illegal XbaI site found at 1680
    Illegal PstI site found at 1035
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2184


Design Notes

Below is an example of a possible implementation of BBa_M36532, the Arsenate Bioremediation Actuator. Due to size limitations, PHO84, ACR 1, and ACR 2, this actuator was created in two constructs, one with the PHO84 (M36533)[1] and another with ACR 1 and 2 (M36534) [2]. In this example, a GAL1 inducible sensor has been attached upstream of both designs. This image shows the different components of the Arsenate Bioremediation Actuator.


Example implementation:

http://i67.tinypic.com/153xj49.png


Glycerol Freezer Stock

Plasmid: ACR2 ACR1

Barcode: 0133027024

Unique ID: HTALE


Plasmid: PHO84

Barcode: 0133029030

Unique ID: PKPHO8411/18/15

Source

DNA 2.0 plasmid backbone (pD1201) with GAL1 inducible promoter, high copy number ORI, and ampicillin and leucine selectable markers.

DNA 2.0 for plasmid backbone (pD1207) with GAL1 inducible promoter, high copy number ORI, and kanamycin and histidine selectable markers. [3]

In order to have a multi-protein expression cassette, we incorporated an Internal Ribosome Entry Site (IRES), which was a T2A sequence. We got the amino acid sequence for this part from the following site [http://blog.addgene.org/plasmids-101-multicistronic-vectors]

Naturally occurring genes in S. cerevisiae: Pho84, ACR 1 and 2 Sequences were referenced from the yeast genome website and then were entered into the registry.

PHO84: BBa_M36535 [4]

ACR 1: BBa_M36536 [5]

ACR 2: BBa_M36537 [6]

References

Engineering of Saccharomyces cerevisiae for Bioremediation of Arsenate and Development of Yeast Vector Tools [http://gradworks.umi.com/35/02/3502760.htm]