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1.3 Detailed description
1.3.1 Abstract
A cell-free system refers to the system of cell environment simulation which consists of cell extract and substrate; it enables DNA expression in vitro. With the help of cell-free system, comprehensive limitations of biochemical reactions, for example, the fatality to the cell due to the toxicity of the product of metabolism and the prospect of the plasmid being controlled by the host due to negative feedback. A cell-free system has relatively higher operability, proved to be a better alternative for DNA transcription and expression. Plasmids assembled via synthetic biological methods can enable cells with the function of biosensors targeting at small molecules (e.g. glucose), hormones(e.g. oestrogen) and oxymethylene. Via adapting these biosensors into cell-free systems via which test papers are made, comprehensive medical detections can be done during everyday life, saving the efforts of comprehensive laboratory tasks. Compared with existing test-paper techniques (e.g. collaurum), the cell-free system has relatively lower manufactural costs while being able to detect molecules in relatively higher quantities. Its functions can be expanded and enhanced via sufficient and efficient application of BioBrick techniques. Based on the theoretical affirmation derived from the research of respectively Keith Pardee on paper-based synthetic gene networks and Alexis Courbet on detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates.
1.3.2 Design
1.3.2.1 Plasmid Design
The genetic circuit is composed of two parts, the sensor device and the amplifier/output device. The design of plasmid had been through three phases (V1-V3) For plasmid composition, see Fig.3.
fig.3 V-1 prototype design Sensor device: The sensor device consists of the promoter for the target molecule and CRE protein expression fragments. It can be activated with via the binding mechanism of small molecules and the promoter(acting as activators or enhancers) or repressed, vice versa, the expression of downstream CRE protein expression. Output device: The output device consists of logic gate component loxp-stop-loxp(loxp fragmentterminator-loxp fragment) and reporter protein (LacZ/GFP/Luciferace,etc.) In the case of CRE expression’s absence, the terminator will stop transcription and translation, and thus preventing the downstream protein’s genetic fragments from being expressed; In the presence of CRE protein expression, the two loxp fragments will cut and reconstruct, resulting in the terminators 180 degree rotation about the plasmid, enabling the expression of downstream reporter genes. If the sensor’s component represses the expression of downstream CRE protein sequences in the presence of transcription factors, we can design the direction of the terminator’s rotation. This design enables analogue signal input’s continuous conversion into digital signal inputs. We have successfully achieved this function during a plasmid test in May using equivalent Boolean logic gate system. For further information, see fig. 4. V2- Output improvements Based on V1, we improved the output plasmid by increasing the number of terminators in the l-s-l region and adding longer CRE protein fragments. Due to the impossibility of ending all transcription
expressions in the plasmid, the terminator’s efficiency can never reach 100%, thus we increased the number of promoters to prevent the distraction of false positive results. The downstream CRE protein sequences will act as amplification device which can cause the rotation of more terminators in the output plasmid, the expression of reporter genes and therefore amplifying the signal via activating a smaller amount of downstream CRE protein expression in the output plasmid in the case of a low transcription factor concentration. V3- Sensor’s improvements To annihilate false positive results, initiate the cutting of output plasmid to prevent its expression via a new genetic editing system, NgAgo/gDNA when the transcription factor(target molecules) are repressing protein expression. NgAgo proteins can cut out a certain 24bp-fragment with the help of a single-strand guide DNA, sgDNA. (Reference. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute by Mr. 韩春雨)
1.3.2.2 The application of cell-free system
We are using the two cell-free systems, S30 and S30T7 based on E.Coli lytic agents provided by Promega.Co. The S30T7 system had a higher transcription expression rate than S30 system as it contains the T7 polymerase, an RNA polymerase which targets at T7 promoters.fig. 4
1.3.2.3 Pearl necklace-shaped test paper design
A concept of semi-quantitative test paper observable by human eye was adapted in previous discussions along with the following instructions: the test paper’s testing zone has a spherical shape, and the concentration of target molecules in the sample is detected by a specific detector containing light source and electronic sensor component. Designed Apps on phones that can initiate the ignition of the flashlight of mobile device was also considered as an alternative. Acknowledging the inactivation effect of cooling on cell-free system and cannot be quantified with certain colour samples although the designs above can provide accurate and articulate test results, an improved test-paper design with the mechanism of paper chromatography was accepted. Prepared cell-free system and plasmids were added and dried in precisely-carved slots. Add specific amount of samples to be tested to the sample region, letting the fluid containing target molecules to flow according to the capillarity mechanism.Each time it passes through a slot, a certain amount of target molecules bind with the plasmid, with the remaining molecules continuing its journey down to the next slot until reaching the completion of reaction. The suitable radius and size of guiding groove will be deduced via modelling, which will also adjust the amount of cell-free system and plasmid added to the paper, resulting in the eventual conversion of number of slots producing reporter proteins into concentration indices.
1.3.3 Methods
1.3.3.1 The application of S30 T7 System
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1.3.3.2 Fragment amplification and plasmid assembly
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1.3.3.3 Creating Estradiol Receptor (E2R) and Progesterone Receptor (PR)
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1.3.3.4 The testing of cell-free system reaction on a paper disc
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1.3.4 Modelling
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