Team:UNITN-Trento/Notebook/Labposts/08/37

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Latest revision as of 09:12, 3 October 2013

{ "date" : "2013-08-15", "author" : "fabio-thomas", "title" : " blue light induction, festivity time!", "content" : " after yesterday disastrous PCR, we decided to take another direction: put the terminator B0015 downstream of j23100_YF1_FixJ, then rebuild the complete device.We started from digestion (biobrick cloning protocol) of 500 ng in 25 ul of both the parts (j23100_YF1_FixJ part starting concentration= 234 ng/ul and B0015 starting concentration = 309,9 ng/ul). We deactivated enzymes, added SAP, deactivated it, then started the ligation: following the protocol, we used concentration values for both parts= 20 ng/ul. We made the control sample and three ligation samples (1:1, 1:2, 1:3); we transformed and plated them.Meanwhile I took back the experiments with the complete device, so I repeated the dilution cultures (5 ml this time) and tried it again, with a Blue lamp and a less intense LED induction. Moreover I induced with blue light the “mask plates” ,with the entire device, to see if colonies produce blue color if induced.Furthermore I inoculate some colonies from Viola’s plate containing the part S04617+EFE.", "tags" : " BlueLight_amilCP-B0015- S04617+EFE " }

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 {
-	"date" : "2013-08-14",
+	"date" : "2013-08-15",
 	"author" : "fabio-thomas",
-	"title" : " blue light induction, NO WAY!!",
+	"title" : " blue light induction, festivity time!",
-	"content" : " <html>  this morning I saw the results of the experiment and… horror: the induced bacteria pellet was lighter then the other two pellets (by the way, they weren’t blue, just a little bit bluish ) THAT SHOULDN’T HAVE HAPPENED!dang it.Maybe the blue LED was too intense, maybe there’s something wrong with our device, don’t know. We only need to take a crack at some other solution at the same time in order to solve this problem: first I will repeat the experiment with less intense blue light, then we are thinking  about cotransforming bacteria with two plasmids, one with j23100-YF1-Fixj, the other with -FixK2-CI-Plambda-amilCPbecause we noticed that the device used yesterday lacks a terminator after fixJ and that could be the reason of the disfunction. Thomas made the cotransformation plates even though the plasmids actually aren’t compatible. Meanwhile we discussed about  using a new backbone for the separate parts so that cotransformation could be achievable: so we made PCR amplification of the new vector pSB4K5, the first part (j23100-YF1-FixJ) and K592020 (FixK2-CI-Plambda-amilCP). As we can see from the gel, the screening of (j23100_YF1_FixJ) didn’t succeed, probably because the backbone’s structure is quite unusual.Furthermore I tried also to plate the entire device on 3 plates in order to make some mask experiment (induce colonies on the plate).</html>",
+	"content" : "<html> after yesterday disastrous PCR, we decided to take another direction: put the terminator B0015 downstream of j23100_YF1_FixJ, then rebuild the complete device.We started from digestion (biobrick cloning protocol) of 500 ng in 25 ul of both the parts (j23100_YF1_FixJ part starting concentration= 234 ng/ul and B0015 starting concentration = 309,9 ng/ul). We deactivated enzymes, added SAP, deactivated it, then started the ligation: following the protocol, we used concentration values for both parts= 20 ng/ul. We made the control sample and three ligation samples (1:1, 1:2, 1:3); we transformed and plated them.Meanwhile I took back the experiments with the complete device, so I repeated the dilution cultures (5 ml this time) and tried it again, with a Blue lamp and a less intense LED induction. Moreover I induced with blue light the “mask plates” ,with the entire device, to see if colonies produce blue color if induced.Furthermore I inoculate some colonies from Viola’s plate containing the part S04617+EFE.</html>",
-	"tags" : " BlueLight_amilCP"
+	"tags" : " BlueLight_amilCP-B0015- S04617+EFE "
 }