Robert W Arnold Week 2
Electronic Lab Notebook
Aipotu Part III: Molecular Biology
- Downloaded Aipotu.
- Spent 10 minutes or so getting used to the program, determining how to identify promoters, terminators, exons, introns, etc.
- Pulled up the DNA comparison between the upper gene window and lower gene window in Green 2.
- Upper gene window was blue, lower gene window was yellow, resulted in a combined color of green.
- Found that bases 79 and 80 were not compatible. Upper gene window had AC in 79 and 80 while the lower sequence had GG.
- Switched base number 68 in the lower gene window from an A to a C and then folded the protein.
- This resulted in the lower gene window producing a white color and the combined color of upper and lower to be blue. Possible sign of blue being dominant over white.
- The difference between green and blue was the 11th amino acid.
- Determined the difference from blue and yellow was the 10th amino acid in each sequence. The sequence in the blue protein had Tyr coded for by TAC while the yellow had Trp coded for by TGG.
- The difference in the red strain also occurred in the 10th amino acid which coded for Phe with TTC. The middle base determines whether blue or red. If it is A, the protein is blue and if it is T, it is red.
- Red and blue combined creates a purple color and yellow and red create an orange.
- Results show incomplete dominance with the combination of color.
- Genetically mutated a strain of red protein by adding in a Tyr with TAC before the Phe. This caused the strand to become purple. Self-crossing this plant will create a pure-breeding purple organism.
- Interestingly, when a Trp was also added to the purple flower sequence, the flower became black.
- White was determined to be the default color in the absence of a fully functioning chain.
- The DNA sequences differed primarily in the range of bases 78 to 83, around the 10th and 11th amino acids. We will use green as the base color. Green had a DNA sequence of 5'-AUGUCUAAUCGGCACAUUUUGUUAGUGUACUGGCGGCAGUAG-3' which coded for a sequence of N-MetSerAsnArgHisIleLeuLeuValTyrTrpArgGln-C. For example, blue and green only differed by one base at base number 83 where a G was replaced by a T. The other starting colors all had this Cys but also had a different bases 78-80 coding for different amino acids. Yellow had TGG coding for Trp, red had Phe coded by TTC, and white had Val coded by GTC.
- No, all white alleles do not have the same sequence. We found 4 or 5 different strains of white throughout our testing of different protein structures. Some different strains we stumbled upon had amino acid sequences of up to 17 or 18 aa long.
- The DNA sequences for all 4 starting colors were identical for the first 9 amino acids of the chain. Green and blue were identical until amino acid 11 where a Cys was switched for a Trp. When compared to green, yellow switch the 10 and 11 amino acid to TrpCys, red switched to PheCys, and the starting white switched out for ValCys.
- In order to create a pure-breeding, we mutated a red protein strain by adding in a Tyr before the Phe at position 10. This caused the strain to become purple. From here, we self-crossed the organism creating a pure-breeding purple organism.
- The changing of the DNA sequence resulted in the changing of colors in each strain. The seemingly minute base changes often caused a radical result in changing color from say green to yellow or to blue. This helped to show that minute changes can have drastic effects to an organism's overall phenotype. Along with this, it was determined that white was the default color for DNA strains that may not be functional due to mutations causing stop codons to be early in the amino acid cycle. This was determined by mutating strains early in the AA cycle.
In today’s lab, DNA sequences were manipulated and mutated to produce different results. The mutations affected flower color and resulted in white, red, orange, yellow, green, blue, purple or black. The template strand we used was a green strand with a DNA sequence of 5'-AUGUCUAAUCGGCACAUUUUGUUAGUGUACUGGCGGCAGUAG-3' and an amino acid sequence of N-MetSerAsnArgHisIleLeuLeuValTyrTrpArgGln-C. From here, the green strain was compared to the other naturally occurring blue, yellow and white strains. It was determined that the differences in the DNA sequence were occurring between bases 78-83, where the 10th and 11th amino acids were being coded. In all of the naturally occurring amino acids, except green, the 11th amino acid was changed to Cys. This Cys in place of Trp caused green to be blue. Then in the 10th amino acid spot, yellow had Trp, red had Phe, and white had Val. While these mutations were seemingly minute (most only involved a changing of 2 base pairs) the effects were drastic to the color of the flower. After this was determined, we began to breed different strains to produce the oranges (red and yellow) and the purples (blue and red). It was also determined that white was the default flower color in the case of a long protein or one shortened by an early stop codon. Then came the process of mutation to produce a pure-breeding purple organism. This was accomplished by adding a Tyr amino acid before the Phe in red. This caused the plant to become purple and then it was self-crossed to produce purple plants. We also found that by adding a Trp before the Tyr in a true purple flower, the flower became black. This assignment was extremely useful. It allowed the students to become familiar with Aipotu, which seems to be a very valuable tool and also refreshed some topics in genetics and molecular biology that may have not been used in a while. The only real unanswered questions after today would be the issue with the introns and exons. A few times, a part of the intron would be changed and it would cause it to not be an intron anymore.
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