Team:UNITN-Trento/Notebook/Labposts/06/63

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(Difference between revisions)

Latest revision as of 07:58, 3 October 2013

{ "date" : "2013-06-27", "author" : "thomas", "title" : "Toxicity test on 5mM Arabinose induced EFE Cells", "content" : "In order to evalutate if the expression of EFE is toxic or not to our cells I created a growth curve. I started from an inoculum of AraCpBAD + EFE that I prepared yesterday. I diluited 50ul of the overnight culture in 5ml of LB and I incubated at 37C with shaker until the cultures reached approximately 0,6 O.D at 600nm. I took 2 samples for negative controls and 2 samples for the induced cells. After that I added 25ul of Arabinose 1M stock solution to the induced samples (5mM working solution) and I registered the O.D. of the samples one time per hour (for 4 hours). In the end I plotted the results.

Plot

As expected, the strong induction of our gene slightly influences on growth-rate (caused by stress) but still is not completely toxic.
To confirm this result I made a toxicity test by serial dilution. I diluited 50ul of overnight culture (one induced sample and one not induced) into 9,5ml of LB. After vortexed the samples, I further diluited them 1:10 for three times. In the end I plated 150ul of each dilution onto Chloramhenicol Plates.

Dilution 1:1000 induced

Dilution 1:1000 not induced

As you can see from the images, there's not big differences in the colony forming ability of the induced and the not induced sample, even in a dilution of 1:1000. Maybe I should dilute them more!", "tags" : "EFE" }

@@ Line 1: / Line 1: @@
 {
-	"date" : "2013-06-24",
+	"date" : "2013-06-27",
-	"author" : "fabio-bruno",
+	"author" : "thomas",
-	"title" : "bacillus subtilis promoters and  plasmids (part 7)!!",
+	"title" : "Toxicity test on 5mM Arabinose induced EFE Cells",
-	"content" : "<html> we realized that the other day we put the wrong antibiotic in the new plasmids inocula; according to that Bruno found out that nothing grew in there. So this afternoon we made inocula again, this time with Ampicilline.We also made some inocula of the 3 confermed promoters just to have enough stock material.</html>",
+	"content" : "<html>In order to evalutate if the expression of EFE is toxic or not to our cells I created a growth curve. I started from an inoculum of AraCpBAD + EFE that I prepared yesterday. I diluited 50ul of the overnight culture in 5ml of LB and I incubated at 37C with shaker until the cultures reached approximately 0,6 O.D at 600nm. I took 2 samples for negative controls and 2 samples for the induced cells. After that I added 25ul of Arabinose 1M stock solution to the induced samples (5mM working solution) and I registered the O.D. of the samples one time per hour (for 4 hours). In the end I plotted the results.<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Plot|<html><center><img src=\"https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png\" style=\"width:450px\"></center></html>}}<html><br/>As expected, the strong induction of our gene slightly influences on growth-rate (caused by stress) but still is not completely toxic. <br/>To confirm this result I made a toxicity test by serial dilution. I diluited 50ul of overnight culture (one induced sample and one not induced) into 9,5ml of LB. After vortexed the samples, I further diluited them 1:10 for three times. In the end I plated 150ul of each dilution onto Chloramhenicol Plates.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Dilution 1:1000 induced|<html><center><img src=\"https://static.igem.org/mediawiki/2013/f/f4/Tn-20130627-Dil1-1000_ind.JPG\" style=\"width:450px\"></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Dilution 1:1000 not induced|<html><center><img src=\"https://static.igem.org/mediawiki/2013/3/30/Tn-20130627-Dil1-1000.JPG\" style=\"width:450px\"></center></html>}}<html><br/>As you can see from the images, there's not big differences in the colony forming ability of the induced and the not induced sample, even in a dilution of 1:1000. Maybe I should dilute them more!</html>",
-	"tags" : " Pveg-PlepA-plasmidPxil-plasmidPspac "
+	"tags" : "EFE"
 }