Difference between revisions of "Part:BBa K1898250"

 
 
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<partinfo>BBa_K1898250 short</partinfo>
 
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BBa_K880005 is used to provide a strong promoter and strong rbs to maximize protein output. The ORF includes GSR, or glutathione reductase that catalyses the conversion of glutathione disulfide to glutathione, and 10x Histidine tag for protein purification. BBa_B0015 consists of a double terminator to end transcription.  
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BBa_K880005 consists of a strong promoter and strong RBS and was used to maximize protein production. This construct codes for GSR (glutathione reductase) and a 10x Histidine-tag. GSR is a catalyst for the conversion of glutathione disulfide to glutathione and 10x Histidine-tag is used for protein purification. BBa__B0015 is a double terminator used to stop transcription.
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<partinfo>BBa_K1898250 parameters</partinfo>
 
<partinfo>BBa_K1898250 parameters</partinfo>
 
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===Sequencing===
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We set up PCR to check if the DNA have the expected bands. We saw the bands at their expected size, which is ~2.1kb.
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The DNA was then sent to sequencing. The sequencing results are unable to confirm the sequence from 854 to 895 bp.
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*The four cutting sites are highlighted in red
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*BBa_K880005 is highlighted in light blue
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*GSR is highlighted in orange
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*10x Histidine-Tag in highlighted in green
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*BBa_B0015 is highlighted in dark blue.
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Sequence with vf2 primer:
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https://static.igem.org/mediawiki/parts/7/7f/Bght_vf2.png
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Sequence with vr primer:
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https://static.igem.org/mediawiki/parts/0/0c/Bght_vr.png
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Although 854 to 895 bp of GSR is not confirmed in the two files provided here, this GSR DNA comes from BBa_K880005. From the sequencing file uploaded in (https://parts.igem.org/Part:BBa_K1898200), 854 to 895 bp of the GSR gene is confirmed to be correct.
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===Protein Gel===
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We ran a protein gel after lysing the CRYAB-HIS construct and just promoter + RBS + CRYAB. The SDS page is shown below:
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https://static.igem.org/mediawiki/parts/e/e2/Cryab_protein_gel.jpeg
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H2O2 causes human CRYAB proteins to aggregate. The expected size for CRYAB (yellow asterisk) is 20 kDa and 21 kDa for CRYAB-HIS (blue asterisk). Adding H2O2 results in a decrease of the original bands at 20 and 21 kDa, and an increase in larger proteins (blue bracket). CRYAB and CRYAB-HIS both increased in size after adding H2O2, suggesting that the addition of a 10x Histidine-tag does not affect the expected behavior for oxidative stress.
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===Protein Purification===
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We wanted to purify the protein using the commercial Capturem His-Tagged Purification Miniprep Kit. There was no protein present in the elutant, so we measured the protein concentration in the flowthrough after lysates were run through the nickel column. The difference between CRYAB-HIS and CRYAB protein concentration in their flowthrough shows that the his tag does bind to the nickel column.
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https://static.igem.org/mediawiki/parts/b/b3/Protein_purification_nanodrop.jpeg

Latest revision as of 12:27, 19 October 2016


Strong promoter + Strong RBS + GSR + 10x Histidine tag + Double terminator

BBa_K880005 consists of a strong promoter and strong RBS and was used to maximize protein production. This construct codes for GSR (glutathione reductase) and a 10x Histidine-tag. GSR is a catalyst for the conversion of glutathione disulfide to glutathione and 10x Histidine-tag is used for protein purification. BBa__B0015 is a double terminator used to stop transcription.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 804
    Illegal BamHI site found at 1488
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 89
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1199
    Illegal SapI.rc site found at 1607



Sequencing

We set up PCR to check if the DNA have the expected bands. We saw the bands at their expected size, which is ~2.1kb. The DNA was then sent to sequencing. The sequencing results are unable to confirm the sequence from 854 to 895 bp.

  • The four cutting sites are highlighted in red
  • BBa_K880005 is highlighted in light blue
  • GSR is highlighted in orange
  • 10x Histidine-Tag in highlighted in green
  • BBa_B0015 is highlighted in dark blue.

Sequence with vf2 primer:

Bght_vf2.png

Sequence with vr primer:

Bght_vr.png

Although 854 to 895 bp of GSR is not confirmed in the two files provided here, this GSR DNA comes from BBa_K880005. From the sequencing file uploaded in (https://parts.igem.org/Part:BBa_K1898200), 854 to 895 bp of the GSR gene is confirmed to be correct.


Protein Gel

We ran a protein gel after lysing the CRYAB-HIS construct and just promoter + RBS + CRYAB. The SDS page is shown below:

Cryab_protein_gel.jpeg

H2O2 causes human CRYAB proteins to aggregate. The expected size for CRYAB (yellow asterisk) is 20 kDa and 21 kDa for CRYAB-HIS (blue asterisk). Adding H2O2 results in a decrease of the original bands at 20 and 21 kDa, and an increase in larger proteins (blue bracket). CRYAB and CRYAB-HIS both increased in size after adding H2O2, suggesting that the addition of a 10x Histidine-tag does not affect the expected behavior for oxidative stress.


Protein Purification

We wanted to purify the protein using the commercial Capturem His-Tagged Purification Miniprep Kit. There was no protein present in the elutant, so we measured the protein concentration in the flowthrough after lysates were run through the nickel column. The difference between CRYAB-HIS and CRYAB protein concentration in their flowthrough shows that the his tag does bind to the nickel column.

Protein_purification_nanodrop.jpeg