Difference between revisions of "Part:BBa K2446055"
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<partinfo>BBa_K2446055 short</partinfo> | <partinfo>BBa_K2446055 short</partinfo> | ||
| − | Engineered Notch have been used in previous project | + | Engineered Notch have been used in previous project [http://2011.igem.org/Team:MIT (iGEM 2011 MIT)]. They replaced the intracellular domain of Notch1 by Gal4-VP16 <partinfo>BBa_K511301</partinfo>. When the extracellular domain of Notch1 binding to the Delta ligand, Gal4-VP64 would be cleavage, located into nuclei, started transcription of its corresponding promoter Gal4-UAS <partinfo>BBa_K511003</partinfo>. |
This year, we took a step forward on their achievement. Referring to a latest publication [[#References|[1]]], we engineered a SynNotch that could recognize surEGFP<partinfo>BBa_K2446051</partinfo> and response with tTa. | This year, we took a step forward on their achievement. Referring to a latest publication [[#References|[1]]], we engineered a SynNotch that could recognize surEGFP<partinfo>BBa_K2446051</partinfo> and response with tTa. | ||
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| − | [[file:T--Fudan-- | + | [[file:T--Fudan--S2.png|800px|middle|''' Figure 3.''': LaG16-SynNotch-tTa can be activated by contacting with surEGFP. Upper image. The rounded cell in lower left corner is K562-surEGFP. In overexposed EGFP view (EGFP/OE), the shadow is a HeLa-LaG16-SynNotch-tTa which express no mCherry, because of noncontact with K562-EGFP. Under image. HeLa-LaG16-SynNotch-tTa expresses mCherry when contacts with K562-surEGFP. The view of slice51 EGFP/OE shows a strong clue that the two kind of cell contact.]] |
<div> | <div> | ||
| − | <B>Figure | + | <B>Figure 2. LaG16-SynNotch-tTa can be activated by contacting with surEGFP. </B>Upper image. The rounded cell in lower left corner is K562-surEGFP. In overexposed EGFP view (EGFP/OE), the shadow is a HeLa-LaG16-SynNotch-tTa which express no mCherry, because of noncontact with K562-EGFP. Under image. HeLa-LaG16-SynNotch-tTa expresses mCherry when contacts with K562-surEGFP. The view of slice51 EGFP/OE shows a strong clue that the two kind of cell contact. |
</div> | </div> | ||
===References=== | ===References=== | ||
[1] L. Morsut et al., Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780--791 (2016). | [1] L. Morsut et al., Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780--791 (2016). | ||
Latest revision as of 20:03, 1 November 2017
SynNotch-anti-EGFP-tTA
Engineered Notch have been used in previous project [http://2011.igem.org/Team:MIT (iGEM 2011 MIT)]. They replaced the intracellular domain of Notch1 by Gal4-VP16 BBa_K511301. When the extracellular domain of Notch1 binding to the Delta ligand, Gal4-VP64 would be cleavage, located into nuclei, started transcription of its corresponding promoter Gal4-UAS BBa_K511003. This year, we took a step forward on their achievement. Referring to a latest publication [1], we engineered a SynNotch that could recognize surEGFPBBa_K2446051 and response with tTa.
Figure 1:SynNotch can be used to recognize surEGFP. HeLa cell stably expresses anti-EGFP synNotch with an activating-form transcription factor (tTa) whose corresponding promoter is pTight. The HeLa cell was infected with a plasmid containing pTight-mCherry. Thus, when LaG16 (an anti-EGFP scFv) binds to surEGFP, Cleavages occurs. tTa will translocate into nucleus and activate pTight. Then mCherry will express.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 381
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2691
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1606
Experiments
In HeLa with LaG16-SynNotch-tTa and pTight cells, the tTa, which originally linked to the intracellular domain of SynNotch, after LaG16 (scFv against GFP) binds to surEGFP, will translocate from the plasma membrane into the nucleus. tTas activate pTight promoter and mCherry starts expression. We used the cells highly expressing surEGFP as the sender. Images show that the HeLa expresses mCherry when contacts with K562-surEGFP, while mCherry won’t be seen in the condition that the HeLa don’t contact with K562-surEGFP. Thus, we confirmed our SynNotch is can respond to surEGFP.
Figure 2. LaG16-SynNotch-tTa can be activated by contacting with surEGFP. Upper image. The rounded cell in lower left corner is K562-surEGFP. In overexposed EGFP view (EGFP/OE), the shadow is a HeLa-LaG16-SynNotch-tTa which express no mCherry, because of noncontact with K562-EGFP. Under image. HeLa-LaG16-SynNotch-tTa expresses mCherry when contacts with K562-surEGFP. The view of slice51 EGFP/OE shows a strong clue that the two kind of cell contact.
References
[1] L. Morsut et al., Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780--791 (2016).