The initial transformation task required two yeast strains (both as Mav203; Leu–, Trp–, His–), with one containing the pDBLeuDa4 bait plasmid, and the other with just the pDBLeu plasmid. The two strains were each transformed with a pPC86DMID1(Y) prey plasmid, where (Y) referred to a full-length positive control (MID1) , a DBB strain (MID1DBB), and a DCC strain (MID1DCC). The transformation process with the two strains and three prey plasmid DNAs resulted in 6 (2x3) transformation mixtures (Table 1).
The transformation mixtures were then spread-plated at 1x and 1/10x dilutions using half of each plate on an SC (synthetic complete) yeast growth medium minus selected amino acid(s); one plate without Leucine (SC-Leu), and two plates without Leucine and Tryptophan (SC-Leu-Trp). Plates were then incubated for 4 days at 30° C, however due to failed growth in the transformation plates for pPC86DMID1 (both yeast strains), and an additional set of results had to be obtained. The spread-plated results for each strain, transformation, result source, and their associated control cases can be seen in Table 2, with a “Yes” growth observation referring to confluent growth and “No” for non-confluent growth.
Following the incubation period, each of the 6 transformation plates were sampled and underwent 4x 10-fold serial dilutions, which were then spot-tested to either an SC-Leu-Trp, or SC-Leu-Trp-His (minus Histidine) medium. Each strain-transformation occupied half of each plate, with a spot per dilution level (Figure 1).
An additional set of spot-tests were also performed to test for auto-activation of the a4 bait protein, using a strain transformed with an unmodified pPC86 plasmid (MaV203+pDBLeuDa4+pPC86).
Following another round of in...
... middle of paper ...
...the DCC variant (for all spot dilution levels). The lack of growth for the DBB variant suggested the B-box region was a requirement for interaction/binding with the a4 protein, while the coiled-coil domain didn’t appear to be a requirement. Its removal however did result in noticeably decreased growth rates when compared to the full-length control test, suggesting its removed resulted in some level of decreased interaction with a4, whether it usually stabilised the binding process, or is removal disrupted the overall form and binding site is unknown, and would require additional testing. However given the external result source, it could have been that group’s sampling and dilution methods had resulted in decreased cell viability, though a simple retest could rule this out, and would be a good starting point to determine any interactions with the coiled-coil domain.
The first step of the experiment was ligation, and the objective was to insert EGFP cDNA into a restriction cut pET41a(+) vector to obtain a recombinant plasmid that would express green fluorescent gene. pET41a(+) was the choice of vector to ligate the EGFP into. Its structural design and genomic sequential properties render it especially well-suited for cloning and high-level expression of peptide sequences. This 5933 bp circular vector contains a built in sequence for Kanamayacin resistance gene. “Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin” (Montserrat, et. al., 2001). This allowed the growth of recombinant and non-recombinant colonies of E. coli, all of which contained the vector insert.
While the previous experiment identified colonies containing recombinant DNA, the patching experiment distinguished which colonies contained the hlyA fragment and which ones did not. Colonies that could cause haemolysis of the blood agar plate indicated that recombinant DNA taken up contained the hlyA fragment ligated with pBluescript, which is the desired subcloning product. The hlyA fragment contains the hlyA gene which encodes for a haemolytic protein that causes the red blood cells in the blood agar to lyse. Therefore, non-haemolytic colonies were transformed with pBluescript plasmid ligated with the pK184 fragment and were not able to cause haemolysis as no hlyA gene was present. In theory, this experiment allowed for the aim to be achieved as it identified colonies with the desired product. Inoculating certain colonies in broth culture allowed for gel electrophoresis to be carried out and confirm if the aim of the experiment has been
Moreover, the class average curve shows a similar trend, as the curve flattens, at 70% but with an enzyme activity of 5.3 x10-3 seconds. This indicates that even though the saturation point is the same it was considerably lower than our results, which could indicate sources of systematic error in the design of the practical.
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
...q DNA polymerase to each tube while disallowing the tubes to cool and without taking
Ligation one was a 1:1 molar ratio pET-41a (+) vector: egfp insert that used 50ng NotI/NcoI cut pET-41a (+) DNA, 7ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Ligation two was a 1:3 molar ratio pET-41a (+) vector: egfp insert made up of 50ng NcoI/NotI cut pET-41a (+), 21ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Water was sterilized and deionized. The remaining three ligation samples served as controls. Ligation three contained 57ng uncut pET-41a (+)/EGFP recombinant plasmid DNA and sterile water. Ligation 4 was a negative control that consisted of only sterile water. Ligation five lacks DNA ligase but has the same properties of the 1:3 molar ratio pET-41a (+)/EGFP vector.
My results did not completely support my hypothesis, while I was correct about pH, temperature, enzyme concentration and inhibitors I was incorrect about substrate concentration. I originally believed that increasing substrate concentration
1. Presence of vertical regenerative walls in both sides of the recipient site or one site which classified as A and B group respectively. Absence of regenerative walls in both side named C group. (Fig.1, 2 ).
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
Schulman, Joshua M., and David E. Fisher. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 28 Aug. 0005. Web. 24 Apr. 2014.
"Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 06 Jan. 2014. .
In the genetic modification of food, a technique called transgenesis is used. It involves incorporating foreign DNA, or desired gene into the organism that is being manipulated. DNA is a long molecule with a double helix structure, present in essentially, all living organisms. It consists of subunits called nucleotides, and has the ability to self-replicate. Organisms that undergo transgenesis are transgenic. A range of techniques is available to transfer genes between organisms. The most common include microinjection and vectors. However, for the genetic modification of food, vectors are the most appropriate method for transgenesis. The most common technique for using vectors is th...
Michener, William K. and Haeuber, Richard A., Bioscience. American Institute of Biological Science. Sep98. Vol. 48. Issue 9. p677.
25 April 2014. Muller, F. Toxicological Sciences. 2014. The 'Standard' of the 'Standard'. 24 April 2014. National Cancer Institute.
There were five test solutions used in this experiment, water being the control, which were mixed with a yeast solution to cause fermentation. A 1ml pipetman was used to measure 1 ml of each of the test solutions and placed them in separated test tubes. The 1 ml pipetman was then used to take 1ml of the yeast solution, and placed 1ml of yeast into the five test tubes all containing 1 ml of the test solutions. A 1ml graduated pipette was placed separately in each of the test tubes and extracted 1ml of the solutions into it. Once the mixture was in the pipette, someone from the group placed a piece of parafilm securely on the open end of the pipette and upon completion removed the top part of the graduated pipette.