Team:UNICAMP-Brazil/Notebooks/September 12
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====PCR: Cre-Recombinase without ATG==== | ====PCR: Cre-Recombinase without ATG==== | ||
- | *<p style=”text-align:justify;”>Today we | + | *<p style=”text-align:justify;”>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.</p> |
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+ | <center> [[Image: Cre_amplificada_copy.jpg]] </center> | ||
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+ | ''Víctor'' | ||
==== 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 | + | *<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> |
- | *<p style=”text-align:justify;”>Today we digested the PY1 and PY2 fragments that have been previously purified with the enzymes | + | *<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 | + | *<p style=”text-align:justify;”>We also digested BBa_J23100 with ''Xba''I and ''Spe''I for 3 hours.</p> |
- | '' | + | ''Fabi and Léo'' |
==''' YeastGuard '''== | ==''' YeastGuard '''== | ||
====New biobricks in biobrick format==== | ====New biobricks in biobrick format==== | ||
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*<p style=”text-align:justify;”>''E. coli'' transformed with the new biobricks grew very well on the LB+AMP plates!</p> | *<p style=”text-align:justify;”>''E. coli'' transformed with the new biobricks grew very well on the LB+AMP plates!</p> | ||
''Taís'' | ''Taís'' | ||
+ | ====RBS problem==== | ||
+ | *<p style=”text-align:justify;”>We did several different 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.</p> | ||
{{:Team:UNICAMP-Brazil/inc_rodape}} | {{:Team:UNICAMP-Brazil/inc_rodape}} |
Latest revision as of 02:29, 22 October 2009
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