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Operon Fusion

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Abstract
Introduction
Methods & Materials
Data & Analysis
Discussion
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Discussion

From the data, the frequency of lysogeny to ampicillin is 1.155 x 10-4 AmpR /pfu and the frequency of lysogeny to arabinose resistance is 7.92 x 10-7 AraR/pFu. The reason there are more ampicillin resistant lysogens than arabinose resistant lysogens is because in order for MC4100 to be ampicillin resistant it only needs to have MuD1 inserted in its genome, regardless of where the lactose structural genes are facing, because MuD1 consists of the Bla gene that is constitutive. However it is much more difficult to have arabinose resistant lysogens because in order for this to occur MC4100 must be carrying the Bla gene and also have a mutation specifically in the arabinose operon.

In order to determine where the MuD1 phage inserted in the arabinose operon one must look at the patched AraR colonies and the most frequently occurring pattern. Unfortunately we did not receive any data for this portion of the experiment, so we cannot determine where the MuD1 phage inserted. We probably failed to infect our cells with the lysate, so when we plated them, nothing would happen because none of them would gain ampicillin resistance from Mud1.  However, if there were white patches on AraMac, LacMac, and Ara+Lac Mac, then it means that Mud1 inserted in either Ara A, Ara B, or Ara C with its lactose structural genes facing away from a promoter. Because MC4100 originally has a point mutation on the Ara D gene, arabinose cannot be used because there is nothing to convert ribulose-5-phosphate into xyulose-5-phosphate in the arabinose metabolic pathway. The build-up of ribulose-5-phosphate is toxic to the cell and the cell will die. However, because the patches were white on AraMac, LacMac, and Ara+Lac Mac this means cells lived and did not use the arabinose or lactose. In order for the cells to live MuD1 must have inserted in either the AraA, AraB or AraC gene because all of these mutations prevent the buildup of ribulose-5-phosphate. The white patches on the LacMac means that the lactose structural genes were not facing a promoter and therefore could not use the lactose, which is how we know MuD1 inserted with LacZ facing away from the promoter. The patch is white on Lac+Ara Mac because it still cannot use arabinose or lactose. If the patches were white on the AraMac and LacMac but red on the Ara+Lac Mac then it means that MuD1 inserted in either AraA or AraB with lactose genes facing an active promoter. The AraMac again has white patches because MC4100 still has its AraD point mutation. We know that Mud1 inserted with its lactose genes facing an active promoter in this case because on Ara+Lac Mac the patches were red, meaning they utilized the lactose. The reason they could utilize the lactose on the Ara+Lac Mac but not on the LacMac is because arabinose and the c gene product is required to turn the promoter on. Since there was no arabinose present in the LacMac, even though the lactose genes were facing the right direction, the promoter was inactive and couldn’t transcribe the lactose genes. If the patches were white on AraMac but red on both LacMac and Ara+Lac Mac then it means that Mud1 inserted in the AraC gene with lactose genes next to the active promoter. A mutation in the AraC gene will make the arabinose operon promoter always on, whether or not arabinose is present, and therefore the lac genes in Mud1 can utilize the lactose with or without the presence of arabinose. Because of this insertion at the AraC gene however, C gene product will not be made so arabinose will not be used.

Based on all three data sets, somewhere between 15% to 22.6% of the MC4100 genome is transcribed on the MacConkey medium. In the selection of Mud1 lysogens there was about 7.5% red colonies in Sue-Lynn’s data, 10% red colonies in Joel and Isleen’s data, and 11.3% red colonies in Alex’s data. On average, 19.2% of the MC4100 genome is transcribed on the MacConkey medium. These red colonies represent Mud1 insertions next to the active promoter. This is one possible orientation of the Mud1 insertion but the other possibility is mud1 inserting with its lactose genes away from the promoter. If we assume that equal amounts of cells had the MudI insertion in both orientations, we double the number of colonies that we know have the insertion. Because of this other possibility there is about between 7.5 x 2= ~15% to 11.3 x 2= ~22.6% of the e.coli genome that is transcribed. A mean of 1.18% of the MC4100 genome is occupied by the araBAD operon. This is calculated by looking at the ratio of arabinose resistant lysogens per pfu to ampicillin resistant lysogens per pfu and multiplying each ratio by 100. Sue-Lynn had .05%, Joel and Isleen had 2.4% and Alex had 1.1% of the MC4100 genome occupied by the araBAD operon.

The control plates tell us about how often spontaneous mutations occur, as opposed to the Mud1 insertion mutations that we cause in the experiment. The spontaneous mutation frequency to ampicillin resistance is less than 5 x 10-9 mutations/vc. The average spontaneous mutation frequency to arabinose is 7.28 x 10-7 mutations/vc for Arar and 2.05 x 10-7 mutations/vc for Ara+ mutations. In terms of interpreting results on AraMac + Amp plates it is good that these frequencies are low. In the experiment we purposefully use Mud1 to regulate mutation so that we can determine where the mutations are occurring. However, if the spontaneous mutation frequencies were high we could no longer use our Mud1 insertions to determine where it inserts because so many mutations would already be occurring, something we could not control in an experiment. For ampicillin resistance, the cell simply needs to be infected by Mud1, which contains the bla gene. For arabinose resistance, Mud1 had to insert into araA, B, or C so that the arabinose metabolism pathway is stopped, allowing the cell to survive in the presence of arabinose. If spontaneous mutation frequency was high we would not know if the cells survived because experimentally induced Mud1 inserted in araA, B or C or if the cell had a spontaneous mutation occur at these 3 genes. For the cell to be wildtype at the arabinose operon, the araD gene must spontaneously mutate back to normal and Mud1 inserted somewhere outside of the arabinose operon. For the araD gene, having a high mutation frequency in the araBAD operon results in more backward mutations in araD so that more cells will survive in the presence of arabinose.

 


Steric Hinderance - Sec 002 - BIOL 302L - FA09