Team:EPF Lausanne/Calendar/10 September 2013

From 2013.igem.org

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{{Template:EPFL2013Header}}
{{Template:EPFL2013Header}}
 +
<font size = "4"> Cell Surface Display</font> <BR>
-
Sensing <BR>
+
"Confocal Microscopy"
 +
<br>- Preparation of all the samples listed the previous day for Immunostaining with FITC conjugated antistreptavidin antibody.
 +
<br>- Preparation of all the samples listed the previous day for fluorescent staining with FITC conjugated biotin.
 +
<br>- Confocal afternoon.
 +
<br><br>
-
'''DpnI digest of the iGEM promoter and the GelE Backbone''' <BR>
+
<font size = "4"> Sensing-Effector </font> <BR>
-
I digested the newly amplified and purified PCR products. The concentrations were not too good but I went ahead with the Gibson Assembly.
+
-
'''Miniprep of the Cad-transformants&PCR thereof''' <BR>
+
''DpnI digest of the iGEM promoter and the GelE Backbone'' <BR>
-
I did the MIniprep of the innoculated cad Transformants and then I did a 20ul reaction PCR in order to check for the presence of the Cad promoter insert. The PCR showed that it was indeed present.
+
-I digested the newly amplified and purified PCR products. The concentrations were not too good but I went ahead with the Gibson Assembly.
-
'''Gibson Assembly of the constitutive promoter+GFP (sensing) and the GelE+GFP construct (effector)''' <BR>
+
''Miniprep of the Cad-transformants&PCR thereof'' <BR>
-
I assembled the constitutive promoter in the plasmid in front of GFP so that we could use it as a positive control for the sensing module.
+
-I did the MIniprep of the inoculated cad Transformants and then I did a 20ul reaction PCR in order to check for the presence of the Cad promoter insert. The PCR showed that it was indeed present.
-
I also inserted the GelE gelatinase gene into the plasmid containing the arabinose sensitve promoter.
+
-
'''Colony PCR of the MMP9 construct''' <BR>
+
''Gibson Assembly of the constitutive promoter+GFP (sensing) and the GelE+GFP construct (effector)'' <BR>
-
In order to check for the presence of the MMP9 insert I did a colony PCR with the colonies that were transformed with this plasmid. But the colony PCR did not work so I decided to innoculate the colonies and do the PCR on the MiniPrep the next day.
+
-I assembled the constitutive promoter in the plasmid in front of GFP so that we could use it as a positive control for the sensing module.
 +
I also inserted the GelE gelatinase gene into the plasmid containing the arabinose sensitive promoter.
-
'''Transformation of cells with the constitutive promoter+GFP and the GelE gelatinase gene''' <BR>
+
''Colony PCR of the MMP9 construct'' <BR>
-
I transformed competent cells with the two last constructs, the one that would serve as a positive control for the sensing module (constitutive Promoter+GFP) and the other one with the gelatinase gene for GelE.
+
-In order to check for the presence of the MMP9 insert I did a colony PCR with the colonies that were transformed with this plasmid. But the colony PCR did not work so I decided to inoculate the colonies and do the PCR on the MiniPrep the next day.
 +
''Transformation of cells with the constitutive promoter+GFP and the GelE gelatinase gene'' <BR>
 +
-I transformed competent cells with the two last constructs, the one that would serve as a positive control for the sensing module (constitutive Promoter+GFP) and the other one with the gelatinase gene for GelE.
 +
 +
'''Note''' <BR>
 +
I also realized that the constructs for the effector module would not express GFP because due to the primers there was a frame shift that induced a stop codon just after the linker and before GFP.
 +
<BR> <BR>
 +
 +
<font size = "4"> Nanoparticles</font> <BR>
 +
 +
''DLS measurements''<BR>
{{Template:EPFL2013Footer}}
{{Template:EPFL2013Footer}}

Latest revision as of 22:19, 4 October 2013

Taxi.Coli: Smart Drug Delivery iGEM EPFL

Header

Cell Surface Display

"Confocal Microscopy"
- Preparation of all the samples listed the previous day for Immunostaining with FITC conjugated antistreptavidin antibody.
- Preparation of all the samples listed the previous day for fluorescent staining with FITC conjugated biotin.
- Confocal afternoon.

Sensing-Effector

DpnI digest of the iGEM promoter and the GelE Backbone
-I digested the newly amplified and purified PCR products. The concentrations were not too good but I went ahead with the Gibson Assembly.

Miniprep of the Cad-transformants&PCR thereof
-I did the MIniprep of the inoculated cad Transformants and then I did a 20ul reaction PCR in order to check for the presence of the Cad promoter insert. The PCR showed that it was indeed present.

Gibson Assembly of the constitutive promoter+GFP (sensing) and the GelE+GFP construct (effector)
-I assembled the constitutive promoter in the plasmid in front of GFP so that we could use it as a positive control for the sensing module. I also inserted the GelE gelatinase gene into the plasmid containing the arabinose sensitive promoter.

Colony PCR of the MMP9 construct
-In order to check for the presence of the MMP9 insert I did a colony PCR with the colonies that were transformed with this plasmid. But the colony PCR did not work so I decided to inoculate the colonies and do the PCR on the MiniPrep the next day.

Transformation of cells with the constitutive promoter+GFP and the GelE gelatinase gene
-I transformed competent cells with the two last constructs, the one that would serve as a positive control for the sensing module (constitutive Promoter+GFP) and the other one with the gelatinase gene for GelE.

Note
I also realized that the constructs for the effector module would not express GFP because due to the primers there was a frame shift that induced a stop codon just after the linker and before GFP.

Nanoparticles

DLS measurements