Difference between revisions of "Team:MIT/Genetic Circuit"
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Above is a diagram of the larger genetic circuit we envision constructing with the individual parts we have characterized. The first component of the circuit (on the far left) checks for characteristics of the disease during the <b>estrogen-high</b> phase of the menstrual cycle and expresses the <b>first recombinase</b> if it senses them. The second component (in the middle) checks for characteristics of the disease during the <b>progesterone-high</b> phase of the cycle and expresses the <b>second recombinase</b> if it senses them. The final component contains the fluorescent output of the circuit - in healthy cells, the transcriptional stop signal between the promoter and the gene, as well as the gene’s being inverted, suppress the expression of yellow fluorescence. In cells with the disease, however, the first recombinase is expressed and cuts out the terminator sequence, and the second recombinase is expressed and inverts the gene to the correct orientation, allowing expression of eYFP. This biological ‘and’ gate with two temporally distinct checks for the disease greatly increases the specificity for an accurate diagnosis. The following walk-through will detail the mechanics of how the circuit works. | Above is a diagram of the larger genetic circuit we envision constructing with the individual parts we have characterized. The first component of the circuit (on the far left) checks for characteristics of the disease during the <b>estrogen-high</b> phase of the menstrual cycle and expresses the <b>first recombinase</b> if it senses them. The second component (in the middle) checks for characteristics of the disease during the <b>progesterone-high</b> phase of the cycle and expresses the <b>second recombinase</b> if it senses them. The final component contains the fluorescent output of the circuit - in healthy cells, the transcriptional stop signal between the promoter and the gene, as well as the gene’s being inverted, suppress the expression of yellow fluorescence. In cells with the disease, however, the first recombinase is expressed and cuts out the terminator sequence, and the second recombinase is expressed and inverts the gene to the correct orientation, allowing expression of eYFP. This biological ‘and’ gate with two temporally distinct checks for the disease greatly increases the specificity for an accurate diagnosis. The following walk-through will detail the mechanics of how the circuit works. | ||
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Revision as of 23:07, 19 October 2016
Genetic Circuit Design
Above is a diagram of the larger genetic circuit we envision constructing with the individual parts we have characterized. The first component of the circuit (on the far left) checks for characteristics of the disease during the estrogen-high phase of the menstrual cycle and expresses the first recombinase if it senses them. The second component (in the middle) checks for characteristics of the disease during the progesterone-high phase of the cycle and expresses the second recombinase if it senses them. The final component contains the fluorescent output of the circuit - in healthy cells, the transcriptional stop signal between the promoter and the gene, as well as the gene’s being inverted, suppress the expression of yellow fluorescence. In cells with the disease, however, the first recombinase is expressed and cuts out the terminator sequence, and the second recombinase is expressed and inverts the gene to the correct orientation, allowing expression of eYFP. This biological ‘and’ gate with two temporally distinct checks for the disease greatly increases the specificity for an accurate diagnosis. The following walk-through will detail the mechanics of how the circuit works.
Healthy cells +Estradiol
In a healthy cell during the estrogen-high phase, miRNA 1 is present in low levels and miRNA 2 is present in high levels. In the first component of the circuit, Repressor 1 is expressed constitutively, and since miRNA 1 is minimally present, this repressor is expressed quite strongly. Despite estrogen’s inducing the pERE promoter controlling Recombinase 1, Repressor 1 and miRNA 1 inhibit production of Recombinase 1. In the second component of the circuit, estrogen induces the production of Repressor 2 under pHybrid. The minCMV promoter controlling Recombinase 2 is constitutive, but Repressor 2 and miRNA 2 are inhibiting production of Recombinase 2. Since neither of the recombinases are being produced, no modifications to the output component occur, and no yellow fluorescence is expressed. Healthy cells +Progesterone
In a healthy cell during the progesterone-high phase, miRNA 1 is still present in low levels, and miRNA 2 is still present in high levels. In the first component of the circuit, the pERE promoter controlling Recombinase 1 is not induced due to the absence of estrogen, so Recombinase 1 is not produced. In the second component of the circuit, progesterone induces pHybrid, causing Repressor 2 to be expressed and, in conjunction with miRNA 2, suppresses the production of Recombinase 2. Again, since neither recombinase is produced, the output component is unchanged, and no yellow fluorescence is expressed. Disease cells +Estradiol
In a diseased cell during the estrogen-high phase, miRNA 1 is up-regulated, and miRNA 2 is down-regulated. Now, in the first component of the circuit, miRNA 1 suppresses the expression of Repressor 1. Since miRNA 2 is no longer present, there is nothing inhibiting production of Recombinase 1 when pERE is induced by estrogen, so the recombinase is expressed and cuts out the transcriptional termination sequence on the output component. In the second component of the circuit, however, estrogen still induces pHybrid and thus production of Repressor 2, so even though miRNA 2 is not present, Repressor 2 still suppresses production of Recombinase 2. Although the transcriptional termination sequence is now gone, the eYFP gene is still inverted, so still no yellow fluorescence is produced.Disease cells +Progesterone
Finally, in a diseased cell in the progesterone-high phase, miRNA 2 is still down-regulated. Although progesterone is present, pHybrid is not induced because the cell is progesterone-resistant - it cannot sense and respond to progesterone. Thus Repressor 2 is not produced, and since miRNA 2 is also not present, Recombinase 2 is expressed, which inverts the eYFP gene to the correct orientation. Even though Recombinase 1 is no longer being produced because estrogen is not present, the transcriptional terminator is already gone because the excision activity of Recombinase 1 is irreversible. Now, when the cell cycles back to the estrogen-high phase, eYFP will be produced, so the cell that the circuit has identified to be diseased is now visibly fluorescing, indicating a positive diagnosis.