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Team:NRP-UEA-Norwich/ComparatorCircuit
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Nitric Oxide Sensing & The Hybrid Promoters | The Comparator Circuit | Theoretical Projects
KHADIJA TO DO
Biological systems function on a great variety of different integrative mechanisms which include post-transcriptional attenuation. We believe that Synthetic Biology is at its most effective when these natural mechanisms are taken and applied in novel situations. This ethos we have sought to emulate by creating our own mechanism of post-transcriptional attentuation; the Comparator Circuit.
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In a previous project, the apparent lack of specificity of the promoter BioBrick we looked to improve upon proved to be a minor difficulty that we felt had not been addressed previously in the Registry. Thus we decided to tackle the issue by devising a way of quantitatively measuring the output of NO with the non-specific promoter we were using through a novel gene regulation system.
Using pairs of BioBricks that result in the complimentary binding of a pair of otherwise standard promoter and reporter constructs, a subtractive effect can be gained to result in altered translation relative to the availability of various substrates within the chassis environment.
The video below describes the project in further detail:
Assembling the various gene constructs was not without its challenges. Due to the fact that complimentary ‘zips’ within the sequence were designed to surround the ribosome binding site it was often the case that the DNA sequence would form unwanted secondary structures that could serve to inhibit the translation of the mRNA in its uncoupled state.
Therefore, when designing the DNA, care was taken to avoid these structures obstructing sequences required for translation of the mRNA. Simultaneously, only common codons for the chassis of interest, E. coli, could be used and these codon had to code for amino acids that were unlikely to change the function of the protein product. This is required since the zip sequences extend past the translational start codon, thus our construct will add a small N-terminal tag to any reporter protein it was attached to.
Using IDT's very helpful online tool, Oligo Analyser, we were able to produce sequences in both constructs required in the Comparator Circuit system that, theoretically, bound together when transcribed at around the same within the chassis, yet left the ribosome bind site open and unfolded when either strand of mRNA lacks its counterpart to complimentarily bind to. The software produced figures demonstrating the likely secondary structure of the constructs made, and their Gibbs Free Energy value at specific temperatures. From this, trial and error eventually resulted in the construction of two BioBricks that have free ribosome binding sites when in isolation, but bind to sequester translation of both mRNAs when bound together (Figure 1).
Due to the stop codon present in the scars of Assembly Standard 10 BioBricks, we decided that our constructs would have to be an Assembly Standard 23 BioBrick. Although the use of Bioscaffolds produced by previous iGEM teams was considered, time constraints meant that changing the Assembly Standard we put our BioBricks in would be the most convenient solution.
. Designed DNA constructs for the subtractive system and had them synthesised
. Made into biobricks (link to registry page)
. Ligating with different promoters and reporters to test
. Ultimately ligate with NO-sensing promoters and effecter enzymes to control NO levels/reporters to detect NO levels quantitatively
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 transcription factor which 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. As the measurement of fluorescence is the output directly related to the rate of translation, the overall equation measures translation, however, transcription is a rate determining process in the gene expression and hence it is factored in here. L/C is the period of time taken for transcription to take place. It is the time in which translation can be initiated but it is unlikely that the two leaders will bind to one another
This part of the equation is the deduction of the knockdown of Construct 1 when there is Construct 2 expression and interaction. The biological constant, k, factors in that not all of construct 2 that is expressed will interact with construct 1 and hence both exist despite, construct 2 existing in small quantities. We believe that depending upon the assembly of the orientation of the two constructs within the plasmid, the interaction and hence efficiency can be altered vastly. If the genes have opposite orientations, so that the termination ends are very close then the reduction of distance will increase the chances of interaction and hence make the sensory system more accurate.
This part of the equation encompasses the natural half life of Construct 1 when it alone is expressed (i.e. no expression or interaction of Construct 2). As described before in the modelling from the basic equation, this is the lower part of the equation and puts it in perspective of Construct 1. The half life is also Construct 1's half life.
So to bring it all together; the top half of the equation indicates the degree of translation of the RNA transcribed by the first promoter under any particular transcription rate of the two promoters in arbitrary units. To make this into a meaningful output it is divided by the maximum translation rate at that rate of transcription to equal E(A1); this indicates the degree of attenuation of one RNA from the other.
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