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Proteins
We generally tend to think of proteins only from a dietary lens, as a component of what we eat. However, they are among the most important and abundant organic macromolecules in the human body, with diverse structures and functions. Every cell contains thousands and thousands of proteins, each with specific functions. Some help in the formation of cellular membrane or walls, some help the cell to move, others act as messages or signals and flow seamlessly from one cell to another, carrying information.
Protein Expression
The method by which living organisms synthesize proteins and further modify and regulate them is called protein expression. Protein expression plays a significant role in several types of research and is highly utilized in molecular biology, biochemistry, and protein research laboratories.
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- MDF57 is a 110kDa DNA binding protein which has some functional roles in the development of lymphomas. In the SDS-PAGE gel, a MW marker is shown followed by the "control" lane, with a sample of recombinant protein "MDF57". Lane 1 shows proteins that have been prepared from leukocytes from a normal, healthy individual. Lanes 2, 3, 4, 5 and 6 show protein prepared from leukocytes from individuals with lymphoma. What is the best explanation for the result shown for patient 3? 250 180 160 140 120 100 60 40 20 MW Marker Control 1 2 Translocatio F57 gene Duplication of MDF57 gene Deletion of MDF57 gene Point mutation of MDF57 gene Normal expression of MDF57 gene 5 |||||||||||| ||||||||||| 6Two approaches for correcting single-gene defects are gene therapy such as is discussed in Section 3-5D and the CRISPR–Cas9 system. Explain why the CRISPR–Cas9 approach can potentially provide more complete restoration of normal tissue function than gene therapy.Describe what each of the 12 bands on your Western blot should have been. Remember, the 12 bands will be 4 conditions x 3 proteins (phospho-S6, phospho-AMPK, tubulin). Please describe the relative density of each band compared to the control (for example, how dense will phospho-S6 be in each of the three experimental conditions compared to the control condition?). For each band, provide a well-reasoned rationale for your anticipated result. Give the reason why cell signaling would produce each result in each condition.
- You are employed in a gene therapy laboratory to test the effectiveness of a vector for correcting the DF508 –CFTR mutation. The vector uses CRISPR –CAS9 genome editing to correct the mutated gene in situ (CFTR-CRISPR). An Ussing chamber experiment was performed using nasal airway cells obtained from a patient with the DF508 CFTR mutation which were transformed with either a control vector consisting of an inert CRISPR-CAS-9 construct (CONTROL) or the active, gene correcting CRISPR CAS-9 construct (CFTR-CRISPR). The following transepithelial voltage values were obtained for the drug regimen shown under open circuit and current clamp (injected current of 10mA) conditions: CONTROL Baseline Amiloride Forskolin Inh172 Vte (mV) -78 -2 -4 -3 DVRt (mV) 8 16 7.5 13 R (W.cm-2) 800 1600 750 1300 I (mA.cm-2) 98 1 5 2 CFTR-CRISPR Vte (mV) -38 -6 -23 -9 DVRt (mV) 9 27 6 11 R (W.cm-2) 900 2700 600…You are employed in a gene therapy laboratory to test the effectiveness of a vector for correcting the DF508 –CFTR mutation. The vector uses CRISPR –CAS9 genome editing to correct the mutated gene in situ (CFTR-CRISPR). An Ussing chamber experiment was performed using nasal airway cells obtained from a patient with the DF508 CFTR mutation which were transformed with either a control vector consisting of an inert CRISPR-CAS-9 construct (CONTROL) or the active, gene correcting CRISPR CAS-9 construct (CFTR-CRISPR). The following transepithelial voltage values were obtained for the drug regimen shown under open circuit and current clamp (injected current of 10mA) conditions: CONTROL Baseline Amiloride Forskolin Inh172 Vte (mV) -78 -2 -4 -3 DVRt (mV) 8 16 7.5 13 R (W.cm-2) 800 1600 750 1300 I (mA.cm-2) 98 1 5 2 CFTR-CRISPR Vte (mV) -38 -6 -23 -9 DVRt (mV) 9 27 6 11 R (W.cm-2) 900 2700 600…You are employed in a gene therapy laboratory to test the effectiveness of a vector for correcting the DF508 –CFTR mutation. The vector uses CRISPR –CAS9 genome editing to correct the mutated gene in situ (CFTR-CRISPR). An Ussing chamber experiment was performed using nasal airway cells obtained from a patient with the DF508 CFTR mutation which were transformed with either a control vector consisting of an inert CRISPR-CAS-9 construct (CONTROL) or the active, gene correcting CRISPR CAS-9 construct (CFTR-CRISPR). The following transepithelial voltage values were obtained for the drug regimen shown under open circuit and current clamp (injected current of 10mA) conditions: CONTROL Baseline Amiloride Forskolin Inh172 Vte (mV) -78 -2 -4 -3 DVRt (mV) 8 16 7.5 13 R (W.cm-2) 800 1600 750 1300 I (mA.cm-2) 98 1 5 2 CFTR-CRISPR Vte (mV) -38 -6 -23 -9 DVRt (mV) 9 27 6 11 R (W.cm-2) 900 2700 600…
- You are employed in a gene therapy laboratory to test the effectiveness of a vector for correcting the DF508 –CFTR mutation. The vector uses CRISPR –CAS9 genome editing to correct the mutated gene in situ (CFTR-CRISPR). An Ussing chamber experiment was performed using nasal airway cells obtained from a patient with the DF508 CFTR mutation which were transformed with either a control vector consisting of an inert CRISPR-CAS-9 construct (CONTROL) or the active, gene correcting CRISPR CAS-9 construct (CFTR-CRISPR). The following transepithelial voltage values were obtained for the drug regimen shown under open circuit and current clamp (injected current of 10mA) conditions: CONTROL Baseline Amiloride Forskolin Inh172 Vte (mV) -78 -2 -4 -3 DVRt (mV) 8 16 7.5 13 R (W.cm-2) 800 1600 750 1300 I (mA.cm-2) 98 1 5 2 CFTR-CRISPR Vte (mV) -38 -6 -23 -9 DVRt (mV) 9 27 6 11 R (W.cm-2) 900 2700 600…You are employed in a gene therapy laboratory to test the effectiveness of a vector for correcting the DF508 –CFTR mutation. The vector uses CRISPR –CAS9 genome editing to correct the mutated gene in situ (CFTR-CRISPR). An Ussing chamber experiment was performed using nasal airway cells obtained from a patient with the DF508 CFTR mutation which were transformed with either a control vector consisting of an inert CRISPR-CAS-9 construct (CONTROL) or the active, gene correcting CRISPR CAS-9 construct (CFTR-CRISPR). The following transepithelial voltage values were obtained for the drug regimen shown under open circuit and current clamp (injected current of 10mA) conditions: CONTROL Baseline Amiloride Forskolin Inh172 Vte (mV) -78 -2 -4 -3 DVRt (mV) 8 16 7.5 13 R (W.cm-2) 800 1600 750 1300 I (mA.cm-2) 98 1 5 2 CFTR-CRISPR Vte (mV) -38 -6 -23 -9 DVRt (mV) 9 27 6 11 R (W.cm-2) 900 2700 600…You are employed in a gene therapy laboratory to test the effectiveness of a vector for correcting the DF508 –CFTR mutation. The vector uses CRISPR –CAS9 genome editing to correct the mutated gene in situ (CFTR-CRISPR). An Ussing chamber experiment was performed using nasal airway cells obtained from a patient with the DF508 CFTR mutation which were transformed with either a control vector consisting of an inert CRISPR-CAS-9 construct (CONTROL) or the active, gene correcting CRISPR CAS-9 construct (CFTR-CRISPR). The following transepithelial voltage values were obtained for the drug regimen shown under open circuit and current clamp (injected current of 10mA) conditions: CONTROL Baseline Amiloride Forskolin Inh172 Vte (mV) -78 -2 -4 -3 DVRt (mV) 8 16 7.5 13 R (W.cm-2) 800 1600 750 1300 I (mA.cm-2) 98 1 5 2 CFTR-CRISPR Vte (mV) -38 -6 -23 -9 DVRt (mV) 9 27 6 11 R (W.cm-2) 900 2700 600…
- 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?In this western blot, the levels of TBK remain constant with increasing amounts/expression of the viral protein (VP). Figure description: Increasing amounts of a plasmid expressing the viral protein (0.5, 1, or 2ug) were cotransfected with TBK1 expression plasmid. Cells were harvested 24 h post-transfection and analyzed for phosphorylated TBK1 (anti-pTBK1 Ser172), total TBK1 (anti-TBK1), B-actin (anti-B-actin), and viral protein (anti-VP) expression by Western blot analysis. VP PTBK1 (S172) TBK1 actin Virus proteins O True O FalseYou are attempting to prepare a single gene knockout library using the pRL27 transposon system. You grow the donor E. coli in Luria broth containing both kanamycin and diaminopimelic acid (DAP) and your recipient Serratia rubidaea in plain Luria broth. You combine an equal ratio of donor and recipient cultures and plate the mixture onto Luria agar supplemented with DAP. After 24 hours incubation at 37°C, you create a cell slurry and plate the cells onto Luria agar aupplemented with kanamycin. After 24 hours incubation at 37°C, you find that no colonies grow. What best explains this outcome? A. Failure to supplement media with DAP B. Failure to remove antiobiotic containing media C. Failure to incubate for a sufficient length of time D. Failure to incubate at the appropiate temperature E. Failure to use the proper mating mix ratio