Team:UNICAMP-Brazil/Notebooks/September 12
From 2009.igem.org
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==== PY Promoter - PY1, PY2 and BBa_J23100 digestion ==== | ==== PY Promoter - PY1, PY2 and BBa_J23100 digestion ==== | ||
- | *<p style=”text-align:justify;”>One of the recognition mechanisms of our project is based on conjugation. In our system, we will use a signal of the beginning of conjugation to stimulate the production of AI-2 in our E.coli. This signal will be a promoter which controls the expression of conjugation-related genes, named PY. (See project overview for more information). | + | *<p style=”text-align:justify;”>One of the recognition mechanisms of our project is based on conjugation. In our system, we will use a signal of the beginning of conjugation to stimulate the production of AI-2 in our E.coli. This signal will be a promoter which controls the expression of conjugation-related genes, named PY. (See project overview for more information).</p> |
- | *<p style=”text-align:justify;”>However, before the construction of this system it is necessary to test the activation of the PY promoter and confirm if it is really activated in the beginning of the conjugation. Therefore we are going to do a test with this promoter. This test consists of constructing a device with this promoter and a RFP reporter, which will indicate when the PY promoter is activated. | + | *<p style=”text-align:justify;”>However, before the construction of this system it is necessary to test the activation of the PY promoter and confirm if it is really activated in the beginning of the conjugation. Therefore we are going to do a test with this promoter. This test consists of constructing a device with this promoter and a RFP reporter, which will indicate when the PY promoter is activated.</p> |
- | *<p style=”text-align:justify;”>We are going to use the 2 sizes of PY amplified from F plasmid: PY1 and PY2 and the part BBa_J23100, which has a promoter (that can be excised with XbaI and SpeI), a RBS, a RFP reporter gene and a terminator. So, we are going to digest the PY1 and PY2 fragments with XbaI and SpeI (the restriction sites of this 2 enzymes were added in the ends of our fragments by the amplification primers) and ligate both of them with BBa_J23100 (previously digested with XbaI and SpeI to remove its promoter). | + | *<p style=”text-align:justify;”>We are going to use the 2 sizes of PY amplified from F plasmid: PY1 and PY2 and the part BBa_J23100, which has a promoter (that can be excised with XbaI and SpeI), a RBS, a RFP reporter gene and a terminator. So, we are going to digest the PY1 and PY2 fragments with XbaI and SpeI (the restriction sites of this 2 enzymes were added in the ends of our fragments by the amplification primers) and ligate both of them with BBa_J23100 (previously digested with XbaI and SpeI to remove its promoter).</p> |
- | *<p style=”text-align:justify;”>Today we digested the PY1 and PY2 fragments (that have been previously purified) with the enzymes XbaI and SpeI. The digestion reaction lasted 3 hours. | + | *<p style=”text-align:justify;”>Today we digested the PY1 and PY2 fragments (that have been previously purified) with the enzymes XbaI and SpeI. The digestion reaction lasted 3 hours.</p> |
- | *<p style=”text-align:justify;”>We also digested BBa_J23100 with XbaI and SpeI for 3 hours. | + | *<p style=”text-align:justify;”>We also digested BBa_J23100 with XbaI and SpeI for 3 hours.</p> |
- | '' | + | ''Fabi and Léo'' |
==''' YeastGuard '''== | ==''' YeastGuard '''== |
Revision as of 23:57, 21 October 2009
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