Difference between revisions of "Part:BBa K2404013:Experience"
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In addition we imaged whole leaves infiltrated with 35S:LUC+ to demonstrate that luciferase expression is easily observable. This allows for the rapid detection of infiltrated leaves without the need for more time consuming quantification. | In addition we imaged whole leaves infiltrated with 35S:LUC+ to demonstrate that luciferase expression is easily observable. This allows for the rapid detection of infiltrated leaves without the need for more time consuming quantification. | ||
Revision as of 16:49, 27 October 2017
Luc+ gene under control of the 35S CaMV promoter
Applications of BBa_K2404013
The part was introduced into agrobacterium using the [http://www.indiana.edu/~pikweb/PDFs%20and%20protocol%20files%20/agrotransform.html freeze-thaw method].
Transformed Agrobacteria were grown o/n at 30C and re-suspended in Induction buffer (10 mM Mes, pH 5.6, 10 mM MgCl2 and 150 μM acetosyringone) to an OD600 of 0.5 or 1.0 and incubated for 2 h at room temperature.
The cultures were infiltrated into expanded leaves of >4 week old Nicotiana benthamiana.
After 3days leaf disks were removed in triplicate from transformed leaves, placed in a tissue-culture dish and luciferin was added to assess luciferase expression.
We varied the experimental parameters in two ways in this assay:
- We used two concentrations of luciferin (0.5mM/1mM)
- We used two concentrations of agrobacteria
For each treatment three leaf disks were removed and the luminescence was measured and then averaged to gain a final value.
Results
We clearly demonstrate that leaf samples infiltrated with the 35S:LUC+ construct show strong luciferase activity.
Therefore this construct is an excellent tool to act as a control for successful tobacco leaf infiltrations.
Previous studies have shown that expression levels of infiltrated proteins can be very variable (Bashandy et al 2015). We also observe this as two leaves with the same treatments showed significant differences in expression (see below Z1 compared to Z2).
Our data show that expression levels do not coincide with the amounts of luciferin or agrobacterium we used in these experiments. Therefore in subsequent experiments we would use 0.5mM luciferin and an agrobacterium concentration of OD600 0.5.
In addition we imaged whole leaves infiltrated with 35S:LUC+ to demonstrate that luciferase expression is easily observable. This allows for the rapid detection of infiltrated leaves without the need for more time consuming quantification.
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