Wet Lab: Part Measurements and Characterisation
In this section you will encounter some different parts with different promoters, and you will perform the measurements at time courses, plot the figures and fit them into the models.
To start with, you will have constitutive promoters with different PoPs strengths.
<bbpart>I20259</bbpart>: Constitutive Promoter 1 (from combinatorial library) – RBS - GFPmut3b - XX
<bbpart>I20260</bbpart>: Constitutive Promoter 2 (from combinatorial library) – RBS - GFPmut3b - XX
Under the individual promoter, green fluorescent protein (GFP) gene is inserted serving as a reporter of the strength of the promoter. You will measure the fluorescence emitted from GFP, and eventually compare the difference among different promoters. Two different ways of measurements will be carried out for each promoter, namely in vivo and in vitro. For in vivo measurement, you will make the measurement directly from cells, while for the in vitro measurement, you will break the cells, spin down the cell debris, and measure the figures from cell lysate supernatant. In this way, you will have a chance to observe the error levels of different measurement methods.
Next you will study the behaviour of an inducible promoter, which will show you how the part can be controlled by a cue coming from outside of the system. Upon the adding of the inducing reagent, you should be able to see a rapid increase of signal.
<bbpart>I7107</bbpart>: pLacI – RBS – GFPmut3b - XX
Finally you will examine a working element: inverter. The element is composed of two parts. One part will control the behaviour of the other: that means when the controlling one increases its level, the level of the other one will decrease.
<bbpart>J45995</bbpart>: Stationary-Phase-Sensitive GFP Generator
<bbpart>J45996</bbpart>: Exponential-Phase-Sensitive GFP Generator
Towards the end of the week, you will be given some purified GFP protein. You will generate a standard curve to convert fluorescent readout into the molecular concentration of GFP.
All in all, you will learn about different DNA parts, the error level associated with measurements, a system controlled by an external trigger, and regulation within a constructed system. In this practical, you will learn how to obtain the experimental data and to convert this data into more meaningful information.