Team:UNICAMP-Brazil/Notebooks/September 12

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*<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;”>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>
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*<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>
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*<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 ''Xba''I and ''Spe''I), a RBS, a RFP reporter gene and a terminator. So, we are going to digest the PY1 and PY2 fragments with ''Xba''I and ''Spe''I (the restriction sites of these 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 ''Xba''I and ''Spe''I to remove its promoter).</p>
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*<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>
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*<p style=”text-align:justify;”>Today we digested the PY1 and PY2 fragments (that have been previously purified) with the enzymes ''Xba''I and ''Spe''I. 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>
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*<p style=”text-align:justify;”>We also digested BBa_J23100 with ''Xba''I and ''Spe''I for 3 hours.</p>
''Fabi and Léo''
''Fabi and Léo''

Revision as of 02:27, 22 October 2009

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ColiGuard

finO+pSB1A3 ligation

  • Since all of our fragments are correctly digested, we started today the ligation of both finO and finP sequences to the vector pSB1A3. Each sequence was ligated singly to the vector and, only after each one is confirmed, we'll join then both to a single construction.

  • We performed the ligation finO+vector pSB1A3 for an O/N period, according to Protocol 11.

Marcelo

finP+pSB1A3 ligation

  • As we did for finO sequence, we also ligated finP to the vector pSB1A3 according to the same protocol. The ligation also lasted O/N period.

Marcelo

PCR: Cre-Recombinase without ATG

  • Today we repeated the PCR reaction in order to isolate the Cre-Recombinase without the ATG start codon. The amplification was succesfully realized, comproved with an 1% agarose gel run. According to the photo, we amplified a fragment of expected size.

Cre amplificada copy.jpg

Víctor

PY Promoter - PY1, PY2 and BBa_J23100 digestion

  • 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).

  • 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.

  • 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 these 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).

  • 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.

  • We also digested BBa_J23100 with XbaI and SpeI for 3 hours.

Fabi and Léo

YeastGuard

New biobricks in biobrick format

E. coli transformed with the new biobricks grew very well on the LB+AMP plates!</p>

Taís

RBS problem

We did several diferent digestions and PCRs with this biobrick but we couldn't confirm its size. The size indicated in Registry doesn't match the sizes we found. Because of it we decide to give up on this biobrick, since our promoters (pDLD and pJEN1) are complete, with the Ribosome Binding Site incluse.




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