Team:UNICAMP-Brazil/Notebooks/October 13

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ColiGuard

Cre-Recombinase - Final Conclusions

Today is October 13th... We intend to already send our assembled biobricks this Thursday (October 15th). We also have less then 10 days to update and finish this entire wiki, and to conclude our mainly experiments.
Therefore, our team decided that we are going to leave aside our Cre's device construction. We already got the part's biobricks, and assembling the entire device will demand a lot of time, and time is kind of lacking right now... =]
It's already a great achievement to successful assemble Cre-Recombinase without it's ATG start codon. So, we are truly satisfied that we actually did it!
Time to work on other stuffs now...

Víctor

PY Promoter - Mini-prep and restriction analysis

Today we performed mini-preps (Protocol 2) to extract the plasmids from the selected colonies inoculated yesterday.

After plasmid extraction we digested them with XbaI and PstI to analyze the sizes of the resulting bands and confirm that our construction is right:

The expected size for the fragment excised with XbaI and SpeI from PY1 + RFP + BBa_J23100 and PY1 + RFP + BBa_B0015 is 1028 bp.

As we can observe in the gel photo, all plasmids presented bands compatible with the expected size for the digestion.

Now that we have confirmed that our construction in biobrick format is right they are ready to be submitted to the registry! =)

We decided to send the PY1 promoter inserted in plasmid BBa_J23100. It is our biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K284008 BBa_K284008]

Fabi and Léo

PCR colony. New device

We found transforming colonies apparently. So, we made colony PCR in order to prove the correct transformation. We used the VF2 and VR primers. We run the agarose gel and???………Yess!!! we get!!!. We observed some samples with band size expected (~840bp)

We selected the samples 2-5-7-19-22. We will make miniprep tomorrow.

Luige, Ane & Marcos

YeastGuard

New Strategy: pGEM

The final confirmation of the lysozyme biobrick was done by digestion of the three plasmids chosen yesterday with EcoRI and PstI. The digestion confirmed two lysozyme biobricks (~600bp of the part and ~3000bp of the vector).

We did miniprep of the promoters’ biobricks and digested the plasmids with EcoRI and PstI to confirm the correct insertion. The digestion of pJEN1 confirmed 1 positive colony (~1000bp) and the pDLD digestion confirmed 2 colonies (~500bp).

We digested the correct biobricks with SpeI and PstI in order to open the vectors and connect the parts. The digestion showed expected fragments (pDLD: 3729bp and pJEN1: 4264bp).

We purified the fragments from the agarose gel and connected them to lysozyme and to the reporter gene YFP, both digested with XbaI and PstI. Then we transformed these ligations in competent E. coli and plated in LB+Amp media.

Raíssa and Taís

@@ Line 4: / Line 4: @@
 ==''' ColiGuard '''==
+====Cre-Recombinase - Final Conclusions====
+*<p style=”text-align:justify;”>Today is October 13th... We intend to already send our assembled biobricks this Thursday (October 15th). We also have less then 10 days to update and finish this entire wiki, and to conclude our mainly experiments.</p>
+*<p style=”text-align:justify;”>Therefore, our team decided that we are going to leave aside our Cre's device construction. We already got the part's biobricks, and assembling the entire device will demand a lot of time, and time is kind of lacking right now... =]</p>
+*<p style=”text-align:justify;”>It's already a great achievement to successful assemble Cre-Recombinase without it's ATG start codon. So, we are truly satisfied that we actually did it!</p>
+*<p style=”text-align:justify;”>Time to work on other stuffs now...</p>
+''Víctor''
 ==== PY Promoter - Mini-prep and restriction analysis ====
@@ Line 17: / Line 26: @@
 *<p style=”text-align:justify;”>As we can observe in the gel photo, all plasmids presented bands compatible with the expected size for the digestion.</p>
-*<p style=”text-align:justify;”>Now that we confirmed that our construction in biobrick format is right they are ready to be sent to MIT! =)</p>
+*<p style=”text-align:justify;”>Now that we have confirmed that our construction in biobrick format is right they are ready to be submitted to the registry! =)</p>
 *<p style=”text-align:justify;”>We decided to send the PY1 promoter inserted in plasmid BBa_J23100. It is our biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K284008 BBa_K284008]</p>
@@ Line 23: / Line 32: @@
 ''Fabi and Léo''
+==== PCR colony. New device ====
-==== PY Promoter - Mini-prep and restriction analysis ====
+We found transforming colonies apparently. So, we made colony PCR in order to prove the correct transformation. We used the VF2 and VR primers.  We run the agarose gel and???………Yess!!! we get!!!. We observed some samples with band size expected (~840bp)
-We found transforming colonies apparently. So, we made colony PCR with in order to prove the correct transformation. We used the VF2 and VR primers.  We run the agarose gel and???………Yess!!! we get!!!. We observed some samples with band size expected (~840bp)