Team:UNICAMP-Brazil/Notebooks/October 16

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__NOTOC__  
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==''' ColiGuard '''==
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==== PY Promoter - Transformation ====
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*<p style=”text-align:justify;”>Today we transformed the conjugative strain with the plasmid with kanamycin resistance containing the RFP reporter under the regulation of PY Promoter. We followed [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3] (Electroporation).</p>
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*<p style=”text-align:justify;”>After the transformation we plated the transformed cells in LB-AMP-KAN plates and let them grow at 37ºC for an O/N period.
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''Fabi and Léo''
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====Yeast experiments====
====Yeast experiments====
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*<p style=”text-align:justify;”>To perform our tests, we digested the following minipreps: pJEN1+YFP, pJEN1+Lys, pDLD+YFP, pDLD+Lys with ''Xba''I and ''Pst''I to connect to YEP vector. We also digested the plasmid YEP+Adh1-YFP to confirm the correct insertion of the part into YEP vector. The digestion showed that our new devices are ricght! =) But there is something wrong with the YEP+Adh1-YFP construction, it seams the digestion were incomplete.</p>
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*<p style=”text-align:justify;”>To perform our tests, we digested the following minipreps: pJEN1+YFP, pJEN1+Lys, pDLD+YFP, pDLD+Lys with ''Xba''I and ''Pst''I to connect to YEP vector. We also digested the plasmid YEP+''ADH1''-YFP to confirm the correct insertion of the part into YEP vector. The digestion showed that our new devices are ricght! =) But there is something wrong with the YEP+''ADH1''-YFP construction, it seams the digestion were incomplete.</p>
[[Image:digested-devices.jpg|600px|center]]
[[Image:digested-devices.jpg|600px|center]]
*<p style=”text-align:justify;”>We purified one digested fragment of each device: pJEN1+YFP (1781bp), pJEN1+Lys(1567bp), pDLD+YFP (1246bp), pDLD+Lys (1032bp). We ligated the digested fragments with YEP vector and transformed in competent ''E. coli''.</p>
*<p style=”text-align:justify;”>We purified one digested fragment of each device: pJEN1+YFP (1781bp), pJEN1+Lys(1567bp), pDLD+YFP (1246bp), pDLD+Lys (1032bp). We ligated the digested fragments with YEP vector and transformed in competent ''E. coli''.</p>
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*<p style=”text-align:justify;”>We did miniprep of YEP+Adh1-lysozyme. Later we digested the plasmids to confirm the insertion of the Adh1-Lysozyme construction into YEP. We also digested 10 more plasmids YEP-Adh1-YFP again, this time we digested a little longer to try to avoid the incomplete digestion problem.</p>
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*<p style=”text-align:justify;”>We did miniprep of YEP+''ADH1''-lysozyme. Later we digested the plasmids to confirm the insertion of the Adh1-Lysozyme construction into YEP. We also digested 10 more plasmids YEP-ADH1-YFP again, this time we digested a little longer to try to avoid the incomplete digestion problem.</p>
[[Image:miniprepYEP+.jpg|400px|center]]
[[Image:miniprepYEP+.jpg|400px|center]]

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ColiGuard

PY Promoter - Transformation

  • Today we transformed the conjugative strain with the plasmid with kanamycin resistance containing the RFP reporter under the regulation of PY Promoter. We followed Protocol 3 (Electroporation).

  • After the transformation we plated the transformed cells in LB-AMP-KAN plates and let them grow at 37ºC for an O/N period.

Fabi and Léo


YeastGuard

Yeast experiments

  • <p style=”text-align:justify;”>To perform our tests, we digested the following minipreps: pJEN1+YFP, pJEN1+Lys, pDLD+YFP, pDLD+Lys with XbaI and PstI to connect to YEP vector. We also digested the plasmid YEP+ADH1-YFP to confirm the correct insertion of the part into YEP vector. The digestion showed that our new devices are ricght! =) But there is something wrong with the YEP+ADH1-YFP construction, it seams the digestion were incomplete.

Digested-devices.jpg

  • We purified one digested fragment of each device: pJEN1+YFP (1781bp), pJEN1+Lys(1567bp), pDLD+YFP (1246bp), pDLD+Lys (1032bp). We ligated the digested fragments with YEP vector and transformed in competent E. coli.

  • We did miniprep of YEP+ADH1-lysozyme. Later we digested the plasmids to confirm the insertion of the Adh1-Lysozyme construction into YEP. We also digested 10 more plasmids YEP-ADH1-YFP again, this time we digested a little longer to try to avoid the incomplete digestion problem.

MiniprepYEP+.jpg

  • We prepares competent S.cerevisiaeand transformed competent yeasts with YEP+Adh1-YFP (Protocol 13) without confirming the correct ligation.

  • We did the YNP Ura- solid medium.

Raíssa and Taís





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