Yeast two-hybrid investigation of Bub1b Introduction The Yeast two-hybrid system was used in this investigation to find which of four DNA inserts codes for a protein able to interact through protein-protein interactions with the protein encoded by the Bub1B gene. The protein encoded by the Bub1B gene is a kinase, which has a role in the formation of the mitotic spindle checkpoint ‘Davenporta et al. (1998)’ Aim • To investigate which of four potential interactor proteins form protein-protein interactions with Bub1B, using a yeast two-hybrid screen and gap-repair cloning. Methods The experimental protocol used in this investigation was as outlined in the GENE223 Lab Manual ‘Gene223 teaching staff (2015)’ in a team of three, using ‘bait’ yeast …show more content…
The class results displayed in Table 2 indicate which of the four ‘prey’ inserts produce proteins able to undergo protein-protein interactions with each strain of Bub1B. A high Leu, Trp, Ade colony count indicates that protein-protein interactions occurred, allowing for yeast growth. From Table 2, BUB3 interacts with strains Bub1B (186-613), Bub1B (324-667) and Bub1B (328-1052). CDC20 interacts with strain Bub1B (186-613). Ppp2rc interacts with Bub1B (328-1052), and Bub1B (588-1052). Zfp207 interacts with no strains of Bub1B, according to Table 2. Saccharomyces cerevisiae cell titre = 1740 x 5 x 1 x 1/10-4 = 1.74x107 …show more content…
This suggests that the orange colony was due to contamination, indicating that there was an error in the experimental procedure used. The team results indicate no protein-protein interaction between the strain Bub1B (328-1052) and any of the four potential interactors. These results differ from those produced by the class for the strain Bub1B (328-1052), which instead indicate protein-protein interactions occurring between the Bub1B protein and BUB3 as well as Bub1B and Ppp2rc, for the strain Bub1B (328-1052). The class Leu, Trp, Ade dropout plates (Table 2) showed that there are interactions between the Bub1B protein produced between 186 and 613 bp on the Bub1B1 gene and CDC20 protein, as shown in Figure 1. There are interactions between the Bub1B protein produced between 328 and 588 bp and BUB3 protein. There are interactions between the Bub1B protein produced between 588 and 1052 bp and Ppp2r5c protein. There are no interactions between the Bub1B and Zfp207
70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first
In B-cells the quality control checks are done with a surrogate light chain to make sure that the heavy chain is functional. During the pro-B-cell stage the heavy chain assembles with the surrogate light chain and Igβ. If it is successful, then it shows it forms a functional pre-B-cell and signals to shut down gene rearrangement at the heavy chain. If it cannot do that then the cell will not get the signal to survive and it will die. Next the B-cell generates a light chain gene diversity in pre-B-cells then is checked for its functional B-cell receptor. Without functional B-cell receptor it will not get the signal to survive and will die.
Since we have already known the amino sequence of the protein in previous step, we can narrow down the targeting ubiquitin ligase based on existing research data such as papers, NCBI data.
Comparing the BLASTx and BLASTp search results, allowed me to determine if I chose the correct ORF in the Toolbox which I believe I did as the proteins found in the BLASTp search were the same as the proteins found in the BLASTx search. The E-values for the same protein found by both the BLASTx and BLASTp searches were also the same but only the start and stop positions of the BLASTx and BLASTp alignments were different for the same protein. Overall, I determined the 5’ UTR to be G1-A28 and the ORF to be A29-G1051.
We tested A type, Alpha type, and mixed type. The key difference between these types are the different genes in Alpha and A type.. These A and alpha type can combine to make a mixed cell via signal transduction pathway. The mixed cells are a combination of A type and Alpha type, these cells can become a haploid, budding haploid, zygote, budding zygote, or a shmoo. A haploid is a single cell. A budding haploid is a single cell with a growth on the side. A zygote is two cells that look like an infinity sign. The budding zygote is 2 cells that are like infinity signs with a growth that looks like a dot. A shmoo looks like a pear, and it the two cells combining together. The mixed type can have shmoos, both haploids, and both zygotes. The A factor and alpha type only have budding haploids and diploids, this is because the A type and Alpha type had nothing to mate with. Single transduction pathways use several steps to produce a cellular response. The yeast cells use G- protein receptors system to mate. G protein receptors are also single transduction pathway. G proteins consist of a signaling molecule, a g protein, G protein coupled receptors and an enzyme. The signaling comes to bind to the G protein on the extracellular side. This causes the G protein coupled receptor to change shape on the cytoplasmic side. When the receptor changes shapes a G protein to bind to it. This activates G
possible error allowing repair thus achieving high fidelity in transcription. Also, the DNA damage response system can activate checkpoints inducing cell cycle arrest, allowing time for different mechanisms such as Base, Nucleotide Excision Repair and Mismatch Repair system which, involving specialized proteins, will excise and repair the incurred error.
Objective The objective of this experiment is to study the effect of varying temperatures on the enzyme catalase by measuring the oxygen production as it breaks down hydrogen peroxide. Introduction Enzymes are used in our daily lives in many ways. From industry to agriculture, enzymes play a necessary role in everything from bread to laundry soap.
During my time in the Brugge Lab, we have utilized our compound screening platform to examine vulnerabilities of cancer cells under therapeutic, environmental, and
The purpose of this lab was to test if yeast could or could not metabolize different types of sugars. The lab can also display how the different types of sugars affect the rate of respiration in yeast. The yeast was tested with each individual sugar to determine the rate of respiration. The smallest sugar had the highest rate of respiration and the largest sugar had the lowest rate of respiration.
BAP1 is a recessive mutant gene with a mild growth defect. The growth defect spread in heterozygous mutants. BAP1 enhances the phenotypes and according to environmental factors it can modulate mutant combinations.
Objective: The purpose of the fermentation lab was to test alcoholic fermentation in yeast. Alcoholic fermentation is the main process that yeast cells use to produce ATP. The objective of the lab was to produce the fluffiest yeast possible through changing the temperature and sugar content with in mixture. The fluffiness is caused by the CO2 bubbles in order to create the ideal mixture of yeast the maximum amount of CO2 is necessary.
al. in 2004 heavily leans on the feedback inhibition model as the major contributor to allelic exclusion in B cells. They studied the effect of phospholipase Cγ1 (PLCγ1), which is expressed throughout B cell development and plays a role in pre-B-cell receptor signaling. When there is reduced expression of PLCγ1 there is impeded early B cell development at the pro-B to pre-B cell transition (Wen et. al., 2004). They also found that Ig heavy chain allelic exclusion was impaired, thus causing defective pre-BCR signaling (Wen et. al., 2004). Rearranged light chains combine with the previously rearranged heavy chain to generate surface IgMs to form the B-cell receptor. This B cell receptor changes the progenitor cell to an immature B
Chaperone proteins in BSE are used to (possibly) refold the misfolded proteins into their correct forms. This may be true because in a normal cell, chaperone proteins promote the correct folding of their substrate proteins by unfolding the incorrect polypeptide chain conformations and providing a sequestered environment
Results: Below are the answers to the given questions about the protein PDB ID: 4EEY.
Discuss in detail the effects that an activating mutation in the IKBKB gene would be expected to have on the downstream signalling pathway components and target gene expression, and how these effects are measured in the article.