Dr CellMaster is studying uptake of the molecule, Burp, by cells which line the

Question

Dr CellMaster is studying uptake of the molecule, Burp, by cells which line the esophagus. You have recently joined her lab and want to WOW your principal investigator. She gives you a dense culture of cells growing in a rich media. You have been instructed to add Burp to the cell culture.
A. Design an experiment to measure the rate that the esophagus cells take up Burp.
B. Design an experiment to determine if Burp enters the cells by passive diffusion, facilitated transport, or active transport.

Dr CellMaster has funding from both NSF and NIH and hence, cost is not an issue.

Explanation / Answer

To design an experiment you must first set a hypothesis, in this case your hypothesis is that the cells has intake of molecule Burp, so for setting variables the independent variable is the one that we are going to study or test (this is the one we are going to isolate)

The independent variable is the one that we are going to study, test or manipulate (this is the one we are going to isolate). In this case, it is the intake of Burpo. The dependent variable is the results of the manipulation of the independent variable (that is, the result of the experiments to be carried out). In this case, to measure the intake of Burp.

It is necessary to know the nature of "burp" if it can be measured by spectrophotometric methods, titration among others. Suppose that this molecule can be measured by a spectrophotometer, for this experiment we put a known "burp" concentration in the medium and take a sample at the beginning of the experiment (sample T0), as time progresses we take samples from the medium every hour (or at reasonable time intervals for the duration of the experiment) until it finishes the experiment. Each sample will be measured in the spectrophotometer and if there is an input of this molecule to the cell then we will see how the molecule disappears from the medium as time progresses. Do not forget the control of this experiment in which it would be the medium without the Burp molecule run in parallel with the main experiment.

To determine the mechanism by which the molecule enters the cell first we must differentiate how it works each type of transport.

Simple diffusion is a non-specific passive transport (small molecules pass through the membranes) which is not mediated by proteins

To determine if there is simple diffusion we can make liposomes from the cells. Once we have the liposomes done we put the liposomes in a medium with "Burp" to measure the entrance of this molecule to the liposomes (using the same scheme as in the previous experiment) In case there is an entrance to the liposomes of the molecule " Burp" will decender its concentration in the external environment and the liposomes will internalize it. The control of this experiment is the liposome placed in the same medium that does not contain the "Burp" molecule

The facilitated diffusion or mediated specific transport is in favor of a concentration gradient, this type of transport is saturable selectio and subject to comptetitive inhibition.

For this experiment we can make proteoliposomes (ie the membranes with the cell proteins) from the cells. Since they do not have a nucleus, these proteoliposomes control their cellular functions, which is why we can assume that they do not produce energy. Using the same approach of the first experiment, we put the proteoliposomes in a medium containing "burp" since there is more Burns in the external environment than in the internal environment until it becomes saturated, once it is saturated we can measure the concentration of "burp" in the external environment, if there was internalization of the molecule then lower the concentration of this molecule of the medium, but we can still measure the concentration of "burp" inside the liposomes and if we find that it is the same then we can assume that it passes through facilitated diffusion.

In active transport it has the same characteristics as the facilitated diffusion with the additional that there is energy expenditure.

To determine if it is active transport we necessarily need the whole cell, and a way to measure its energectic level (ie ATP co-concentration, or some other high-energy molecule) We put the cell in the "burp" presence and if there is energy expenditure then the concentration of ATP will decrease, the control for this experiment would be to put the cell in the same medium without Brup and see that their ATP levels are maintained, but even a negative control would be an experiment in which instead of adding "burp" "in the middle we put an analago of" Burp "which blocks the protein through which it enters so that once this analago reaches the receptor the energy level of the cell will remain the same.

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