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Team:NRP-UEA-Norwich/Week10
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Welcome to the NRP UEA iGEM 2012 Wiki Lab Book
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Contents |
. As a backup, the running some of the running culture of the Comparator circuit 1 and 2 transformed cells were mini-prepped. After nanodropping, the concentration was found to be:
C1-1a: 733.4 ng/µL
C1-2a: 1192.6ng/µL
C1-3a: 1596.9 ng/µL
C1-4a: 854.8 ng/µL
C2-1a: 896 ng/µL
C2-2a: 712.5 ng/µL
C2-3a: 1196.7 ng/µL
C2-4a: 811.9 ng/µL
. Following this an overnight double restriction digest with EcoR1 and Pst1 was set up. These were set up using: 0.2µl BSA, 2µl Buffer H, 0.5µl EcoR1 and 0.5µl Pst1. As to the DNA, 1µg of DNA was added:
C1-1a: 1.36 µL
C1-2a: 0.84 µL
C1-3a: 0.62 µL
C1-4a: 1.17 µL
C2-1a: 1.12 µL
C2-2a: 1.40 µL
C2-3a: 0.84 µL
C2-4a: 1.23 µL
. Following a low concentration reading on Friday, B-M was miniprepped again. However, this produced no DNA when nanodropped. Therefore, B-M was reinoculated into culture.
. M-B samples were digested with Spe1 and Pst1 to linearise the backbone to ligate a reporter (CFP, or RFP) into it. The small fragment that was released was discarded during gel purification. At the same time CFP and RFP were digested with Pst1 and Xba1 and then gel purified ready for ligation with M-B. The restriction digests involved 0.5µL of each enzyme, 2µL of Buffer B (Spe1 and Pst1)and Buffer H (Pst1 and Xba1), 0.2µL of BSA. Again 1µg of DNA was used. The gel used to separate the fragments was 1% w/v.
. Multiple ligations were set up: MB2, MB3,MB5,MB9,MB10 were set up to ligate with both RFP and CFP and Comparator circuit 1 and 2 (all samples) were set up to be ligated in the iGEM backbone pSB1C3. These were all left overnight.
. The ligations (MB with CFP/RFP and Comparator Circuit 1 and 2 with pSB1C3) from the night before were run on a 1% w/v gel for an hour and the fragments were cut out. These gel slices were purified. The purified DNA, was then transformed into Bioline Alpha Select Gold Standard Competence cells. The plates were left overnight in an incubator at 37 degrees Celsius.
. Samples of B-M were miniprepped and nanodropped. The concentrations were found to all be lower than 45ng/L. Furthermore, the 260/230 readings were all negative.
. Digested:
working mathmatical modelling
E = the expression of one of the fluorescent protiens (RFP) when there is transcription of the CFP RNA at any particular level as a proportion of the expression of RFP at the same transcription rate when none of the CFP RNA is present within the cell. So if the promoter (PYEAR) attached to the rfp and construct 1 was expressing at a constant rate with promoter 2 entirely switched off then promoter 2 started transcribing and the amount of rfp in the cell halved then the value of EA would be 0.5 at that transcription rate of CFP (0-1)
L = the length of the DNA strand that is transcribed (Leader and protein coding region) .
C = the rate of transcription
L/C = the period of time taken for transcription to take place, the time in which translation can be initiated but it is unlikely that the two leaders will bind to one another
A = the rate of transcription of promoter 1 (the PYEAR) as a proportion of it’s maximum possible transcription rate (0-1)
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.
H = Half life of the RNA after transcription
L/C + H = the full time for which the RNA would be translated assuming no interactions between leaders for instance when only one of the promoters is inducing transcription.
B = the transcription of the second promoter within the cell as a proportion of the maximum possible transcription of that promoter (0-1)
Because there can not be negative expression of A (only positive expression of B to represent negative expression of A) the translation of the RFP RNA(A) = A - AB .
It also has to be remembered that there will never be full interaction between the two RNA leaders, particularly at low concentrations if only because the two strands never come in to proximity or because of cellular processes; consequently the function B/(D+B) must be used giving the formula; translation of the RFP RNA(A) = A – A (B/(D+B))
D is a constant of the biological system whose derivation is so complex that it can only really be calculated through observation but can be modelled at various levels.
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 EA ; this indicates the degree of attenuation of one RNA from the other.
To get the degree of translation of the other RNA (EB) just swap A for B throughout the equation.
. A B-M overnight culture was miniprepped by Russell, and nanodropped. The concentration was found to be: 199 ng/µL
. This was then digested with Spe1 and Pst1. Again 1µg of DNA, 0.5µL of each of Spe1 and Pst1, 0.2µL BSA and 2µL Buffer B.
. This was then run on a 1% w/v agarose gel for an hour and the large fragment purified.
. Multiple overnight ligations were set up for BM1 and BM4 with RFP and CFP. These were left in the dark.
. All the plates which showed growth were inoculated into 5ml LB cultures containing the relevant antibiotics.
. As back up, Comparator circuit 1 and 2 was transformed again and plated. These plates were grown overnight at 37 degrees Celsius.
