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Welcome to the NRP UEA iGEM 2012 Wiki Lab Book
Please choose the relevant link to access our diary of that week!
Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments
The idea of the comparator circuit is to provide a modular sensor which can specifically and quantitatively measure different chemical species within the cell. Through theory, an equation has been assembled which can measure the expression of, in the characterisation stages, reporter proteins such as RFP and CFP.
Figure: Theoretical equation to measure the difference between expression levels of Construct 1 and 2. For explanation reasons, the full equation has been laid out in a way that is relative only to Construct 1, the numbers can be reversed to be relative to Construct 2.
E = Proportion of rate of expression of Construct 1 when both constructs are expressed (i.e. there is knockdown of one construct) relative to the expression of Construct 1 when only Construct 1 is expressed.
A = The rate of transcription of Construct 1 as a proportion of the maximum transcription rate. As a proportion this is measured on a scale of 0 - 1. As an example if the rate of transcription is half of the maximum rate, rate would be 0.5 (arbituary units). It can be assumed the rate of transcription of construct 1 and 2 due to cellular components (e.g. RNA polymerase) is the same, however, the activation of transcription will affect the rate. The activation is reliant on the chemical species interacting with the promoter (i.e. nitric oxide,nitrates,nitrites to PyeaR). The '1' and '2' refer to the Construct 1 or 2 and hence the promoter and the measured fluorescence protein attached (e.g GFP, RFP, CFP, etc).
L = The length of the Construct 1 in the DNA form that is transcribed (i.e the leader and protein coding region).
Note: Leader refers to the section of RNA at the start of the mRNA that is not translated but has an affect on translation rate.
C = The rate of transcription. Assuming the rate of transcription of Construct 1 and 2 are the same because the same ribosomes and RBS are involved.
T = Half life of Construct 1 when only Construct 1 is present; the natural half life of Construct 1.
K = A constant of the biological system. This can only be measured through observation.
The full equation is modeled on the basic equation of:
where E is the rate of expression and E(A1) is the same as that explained above.
The additional complexity factors in less assumptions, and inaccuracies. Below is a breakdown of the full equation.
This refers to the translation of Construct 1 when there is no Construct 2 to knock down Construct 1. The length of DNA is particularly important when the chassis is bacterial. In bacteria, as there is no true nucleus, translation occurs simultaneously with transcription. This affects the probability of interaction between construct 1 and 2 before they are translated. The rate of transcription of construct 1 is important to the expression of construct 1 because
A*(L/C) = the number of RFP RNAs that can be translated independently of the presence of other RNA at any one time and so is proportional to translation (and expression) from DNA ascociated RNA (RNA that is still being transcribed). This occurs both when the CFP RNA is and isn’t present so has to be on both the top and bottom of the equation.
. Paragraphs on the breakdown of the equation
. Importance of parts
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