Difference between revisions of "Part:BBa K2407000"
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<partinfo>BBa_K2407000 short</partinfo>
 
<partinfo>BBa_K2407000 short</partinfo>
  
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<h3>Design</h3>
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<hr>
 
<p>This is a shortened <i>CUP1</i> promoter, improved from BBa_K2165004 (<partinfo>BBa_K2165004</partinfo>). This promoter is designed to be deleted with a few bases on the two ends. We retained 5 <i>ACE1/CUP1</i> binding sites, 2 TATA boxes, and one initiation element in the promoter. The complex of <i>ACE1</i> and copper ions will bind the promoter, which causes the activation of <i>CUP1</i> promoter with TATA boxes' help. In this way, this promoter played its key role with less bases.</p>
 
<p>This is a shortened <i>CUP1</i> promoter, improved from BBa_K2165004 (<partinfo>BBa_K2165004</partinfo>). This promoter is designed to be deleted with a few bases on the two ends. We retained 5 <i>ACE1/CUP1</i> binding sites, 2 TATA boxes, and one initiation element in the promoter. The complex of <i>ACE1</i> and copper ions will bind the promoter, which causes the activation of <i>CUP1</i> promoter with TATA boxes' help. In this way, this promoter played its key role with less bases.</p>
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[[File:17Tianjin Redesigned CUP1 promoter.png|700px|thumb|center|'''Fig 1'''.Structure of redesigned CUP1 promoter used in our project, based on BBa_K2165004]]
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<h3>Characterization</h3>
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<hr>
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<p>Strains of <i>S. cerevisiae BY4742</i> containing either BBa_K2165004-yEmRFP and BBa_K2407000-yEmRFP with an initial OD600 of 0.1 were grown for 24 hours in SC-URA medium at 30 degrees Celsius, and then were induced with 0.1 mM Cu2+. Samples were tested with fluorescent microplate reader after 1, 3, 6, 12, and 24 hours.</p>
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[[File:|700px|thumb|center|'''Fig 2'''.The characterization of both BBa_K2165004 and BBa_K2407000]]
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<p>Figure 2 shows the expression of yEmRFP with both the two promoters were very similar, so we can tell the deletion didn’t influence the core function of CUP1 promoter. Based on the new part, we carried out a further improvement.</p>
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<h3>Further Improvement</h3>
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<hr>
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<p>The technology of Error-Prone PCR was taken into our experiment to reduce the leakage expression and increase the response rage. Visist <partinfo>BBa_K2407013</partinfo>, <partinfo>BBa_K2407014</partinfo>, <partinfo>BBa_K2407015</partinfo>, and <partinfo>BBa_K2407016</partinfo>.
  
 
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Revision as of 11:34, 27 October 2017


Shortened CUP1 promoter without RFC sites


Design

This is a shortened CUP1 promoter, improved from BBa_K2165004 (BBa_K2165004). This promoter is designed to be deleted with a few bases on the two ends. We retained 5 ACE1/CUP1 binding sites, 2 TATA boxes, and one initiation element in the promoter. The complex of ACE1 and copper ions will bind the promoter, which causes the activation of CUP1 promoter with TATA boxes' help. In this way, this promoter played its key role with less bases.

Fig 1.Structure of redesigned CUP1 promoter used in our project, based on BBa_K2165004

Characterization

Strains of S. cerevisiae BY4742 containing either BBa_K2165004-yEmRFP and BBa_K2407000-yEmRFP with an initial OD600 of 0.1 were grown for 24 hours in SC-URA medium at 30 degrees Celsius, and then were induced with 0.1 mM Cu2+. Samples were tested with fluorescent microplate reader after 1, 3, 6, 12, and 24 hours.

[[File:|700px|thumb|center|Fig 2.The characterization of both BBa_K2165004 and BBa_K2407000]]

Figure 2 shows the expression of yEmRFP with both the two promoters were very similar, so we can tell the deletion didn’t influence the core function of CUP1 promoter. Based on the new part, we carried out a further improvement.

Further Improvement

The technology of Error-Prone PCR was taken into our experiment to reduce the leakage expression and increase the response rage. Visist BBa_K2407013, BBa_K2407014, BBa_K2407015, and BBa_K2407016. Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]