Genome-wide RNAi screens target expression of > 16,000 genes. Explain how each of these 16,000+ bacterial strains would be engineered in order that they only cause gene silencing of the intended target.
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Bacterial Genomics
The study of the morphological, physiological, and evolutionary aspects of the bacterial genome is referred to as bacterial genomics. This subdisciplinary field aids in understanding how genes are assembled into genomes. Further, bacterial or microbial genomics has helped researchers in understanding the pathogenicity of bacteria and other microbes.
Transformation Experiment in Bacteria
In the discovery of genetic material, the experiment conducted by Frederick Griffith on Streptococcus pneumonia proved to be a stepping stone.
Plasmids and Vectors
The DNA molecule that exists in a circular shape and is smaller in size which is capable of its replication is called Plasmids. In other words, it is called extra-chromosomal plasmid DNA. Vectors are the molecule which is capable of carrying genetic material which can be transferred into another cell and further carry out replication and expression. Plasmids can act as vectors.
Genome-wide RNAi screens target expression of > 16,000 genes. Explain how each of these 16,000+ bacterial strains would be engineered in order that they only cause gene silencing of the intended target.
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- Given the following Western blot results showing levels of proteins over time in three yeast cell extracts after the induction of high level expression of the CDH1 protein. WT GAL-CDH1 GAL-CDH1, cdc23-1 0 30 60 120 0 30 60 120 0 30 60 120 Time (min) - Pds1 - Clb2 - Ase1 - Kar2 Note: The GAL-CDH1 indicates that the yeast strain contains a transgenic cdh1 gene under the control of the GAL promoter. This means that the researcher can control expression of that cdh1 gene. In these three experiments (three yeast strains), time 0 is the point at which GAL- controlled genes begin to get expressed (WT lacks transgenes). In this experiment, four proteins are being probed for in three different yeast strains (1. wild-type, 2. a strain containing the cdh1 gene under GAL control (see note), and 3. the GAL-CDH1 strain that is also harboring a cdc23 loss of function allele (allele 1), which is a component of the A The upper (larger) band in the Ase1 lanes is non-specific background band; disregard…Analyzing Cloned Sequences A base change (A to T) is the mutational event that created the mutant sickle cell anemia allele of beta globin. This mutation destroys an MstII restriction site normally present in the beta globin gene. This difference between the normal allele and the mutant allele can be detected with Southern blotting. Using a labeled beta globin gene as a probe, what differences would you expect to see for a Southern blot of the normal beta globin gene and the mutant sickle cell gene?Given the following Western blot results showing levels of proteins over time in three yeast cell extracts after the induction of high level expression of the CDH1 protein. WT GAL-CDH1 GAL-CDH1, cdc23-1 0 30 60 120 0 30 60 120 0 30 60 120 Time (min) | Pds1 - Clb2 - Ase1 - Kar2 Note: The GAL-CDH1 indicates that the yeast strain contains a transgenic cdh1 gene under the control of the GAL promoter. This means that the researcher can control expression of that cdh1 gene. In these three experiments (three yeast strains), time O is the point at which GAL- controlled genes begin to get expressed (WT lacks transgenes). which is a component of the A In this experiment, four proteins are being probed for in three different yeast strains (1. wild-type, 2. a strain containing the cdh1 gene under GAL control (see note), and 3. the GAL-CDH1 strain that is also harboring a cdc23 loss of function allele (allele The upper (larger) band in the Ase1 lanes is non-specific background band; disregard it.…
- a) The best vector to use determine receptor binding protein expression would have been one with a GFP gene (green flourescent protein) attached. State one (1) reason why including this gene would have made the experiment easier and whether you have inserted the receptor binding domain gene before or after the GFP gene. b) You wish to determine the sucess pf your transformation by detecting the presence of receptor binding domian mRNA. Describe the key steps of the hybrdization technique you would use, clearly stating how you would design the probe to detect your receptor mRNA.We are utilizing BL21 DE3 bacterial cells for the expression of the ADA protein via autoinduction. Create a schematic/figure showing the biological mechanism for expressing our desired protein in this cell line. We use the DE3 lysogen for expressing T7 polymerase and our plasmid has kanamycin resistance (not ampicillin).Create a schematic of this expression.Suppose you have six strains of E. coli. One is wildtype, and each of the other five has a single one of thefollowing mutations: lacZ−, lacY−, lacI−, oc, andlacIS. For each of these six strains, describe thephenotype you would observe using the following assays. [Notes: (1) IPTG is a colorless synthetic molecule that acts as an inducer of lac operon expressionbut cannot serve as a carbon source for bacterialgrowth because it cannot be cleaved byβ-galactosidase; (2) X-gal cannot serve as a carbonsource for growth; (3) E. coli requires active lactosepermease (the product of lacY) to allow lactose,X-gal, or IPTG into the cells.] Colony color in medium containing glycerol as theonly carbon source and X-gal, but no IPTG.d. Colony color in medium containing high levels ofglucose as the only carbon source, X-gal, andIPTG.e. Colony color in medium containing high levels ofglucose as the only carbon source and X-gal, butno IPTG
- Answer as Directed. Below is the model of a lac operon. lac I lac Z с promoter operator +1 lac Y lac A DNA 1. In the absence of lactose and the presence of glucose in the bacterial growth media, what proteins are bound to the lac control region? Is the operon being transcribed then? 2. In the presence of lactose and the presence of glucose in the bacterial growth media, what proteins are bound to the lac regulatory region? Is the operon being transcribed then? 3. In the presence of lactose and the absence of glucose in the bacterial growth media, what proteins are bound to the lac control region? 4. Why is it adaptive for a bacterium to not express the genes that encode for that lactose utilization proteins when lactose is not available or when glucose is present? 5. Why is it adaptive for the structural genes for using lactose to be under the control of a single promoter, i.e., synthesize a polycistronic message rather than three monocistronic message?explain please: Spike-Driven Syncytia Formation Is Coupled to S20 Fragment Generation in the Presence of ACE2. To investigate the functional and biochemical signatures of spike (S) protein upon engaging its receptor ACE2 in live cells, we first utilized a cell–cell fusion assay to obtain potentially cleaved spike protein products from syncytia . In this system, HEK293T cells transfected with plasmids encoding WT spike readily formed syncytia after coculturing with HEK293T cells expressing human and Vero E6-ACE2, as well as colorectal adenocarcinoma Caco-2 and lung adenocarcinoma Calu-3 cells expressing endogenous ACE2. We then collected these adherent syncytia and immunoblotted for cleaved spike species using a rabbit polyclonal antibody specifically detecting the S2 ectodomain through standard reducing Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis. This experiment revealed that spike expression in HEK293T cells displayed full-length S and autocleaved S2…The streptolysin S toxin made by S. pyogenes is encoded by a 9-gene operon, sagABCDEFGHI. Thinking about what a 3-line diagram would look like for this operon, answer the following questions. Write numeric answers only. For example, if your answer is 6 promoters, write only 6. 1) How many promoters control the expression of these genes? 2) How many locations does RNA Polymerase bind to get full expression of these genes? 3) How many ribosome binding sites are needed for full protein expression? 4) How many start codons will be needed for full protein expression? 5) How many mRNA strands will be produced with full operon expression? 6) How many proteins will be produced with full protein expression? 1
- A number of mutations affect the expression of the lac operon in E. coli. The genotypes of several E. coli strains are shown below. ("+" indicates a wild-type gene with normal function and "-" indicates a loss-of-function allele.) Please predict which of the following strains would have the lowest beta-galactosidase enzyme activity, when grown in the lactose medium. OF POZY Ort Ptot Z¹ Yt Ort p²o+z¹Y+ Orpt ot ztyFor each of the E. coli strains that follow, indicate theeffect of the genotype on the expression of the trpEand trpC genes in the presence or absence of tryptophan. [In the wild type (R+ P+ o+ att+ trpE+ trpC+),trpC and trpE are fully repressed in the presence oftryptophan and are fully expressed in the absence oftryptophan.]R = repressor gene; Rnproduct cannot bind tryptophan; R− product cannot bind operatoro = operator for the trp operon; o− cannot bind repressoratt = attenuator; att− is a deletion of the attenuatorP = promoter; P− is a deletion of the trp operonpromotertrpE− and trpC− are null (loss-of-function) mutationsa. R+ P− o+ att+ trpE+ trpC+b. R− P+ o+ att+ trpE+ trpC+c. RnP+ o+ att+ trpE+ trpC+d. R− P+ o+ att− trpE+ trpC+e. R+ P+ o− att+ trpE+ trpC−/R− P+ o+ att+trpE− trpC+f. R+ P− o+ att+ trpE+ trpC−/R− P+ o+ att+trpE− trpC+g. R+ P+ o− att− trpE+ trpC−/R− P+ o− att+trpE− trpC+Explain why RNAi would be a less effi cient mechanism for regulating the expression of specifi c genes if Dicer hydrolyzed double-stranded RNA every 11 bp rather than every 22 bp.