IPTG_09-10 1IPTG_09-10 2APh 162 – Cellular Decision Making Measuring Gene Expression Winter 2010 Introduction: If you had to choose between broccoli and chocolate, would you eat both simultaneously or one at a time? Which one would you eat first? How and what do you think a mouse or a fly would choose? What about a bacterium? According to the famous Monod’s experiment1, E. coli can choose to eat a certain sugar even when multiple types of sugar are present in the growth media. Today, we are going to focus on one of the most well known examples—the lac operon. The lac operon controls the production of three different genes associated with the metabolism of lactose. One of these genes is lacZ, which encodes for the enzyme beta-galactosidase which metabolizes lactose. When the growth media has glucose and lactose E. coli will not produce any beta-galactosidase. It will only produce it once it has consumed all the glucose. This means that the cell has a way of determining if there is no glucose and if there is lactose in the media. In this experiment we will explore the part of the circuit that takes care of detecting the presence or absence of lactose in the media. Instead of lactose, which would get eventually eaten by the cells, we use the inducer IPTG. This small molecule interacts with Lac repressor in much the same way that the real sugar does except that it cannot be cleaved by the enzyme beta-galactosidase, making it a useful experimental substrate. In the absence of lactose or IPTG, Lac repressor binds to an operator that overlaps the lac promoter. Because of this overlap, RNA polymerase cannot bind to DNA to initiate transcription, resulting in repression. More interestingly, there are three operators in the wild type lac operon with different binding strengths. Tetramerized Lac repressor can even bind to two operators at the same time, resulting in a DNA loop which enhances the repression effect. Fig 1. Repression in the lac operon. (A) A RNA polymerase molecule binds to its promoter on the DNA to initiate transcription. (B) When a repressor molecule binds to its operator, it blocks part of the promoter and leads to repression. (C) A tetramerized repressor can bind to two operators at the same time, thus forming a DNA loop and enhancing the repression effect. 1 Jacques Monod "The growth of bacterial cultures" Annual Reviews Microbiology. 1949. 3:371-394. A. B. C.However, no response in biology is really digital. There is no such thing as something being “on” or “off”. You can instead ask an analog question: to what extent is something off? The Construct: In this experiment, we will use two synthetic sequences for the lac operon: one with a single operator O1, and another one with two operators Oid (an operator with ideally strong binding strength) and O1. In both of the sequences, the target lacZ gene was replaced by a YFP gene controlled by a promoter called lacUV5 (a mutant of the wild type lac promoter). Fig 2. The construct. (A) Sequence with a single operator O1. (B) Sequence with two operators: Oid and O1. The length of the spacer DNA between the two operators is 72.5bp. The promoter in both of the sequences is lacUV5 controlling the expression of a YFP gene. Measuring gene expression: We will measure the fluorescence expressed by cells containing the construct described above for different concentrations of IPTG. We want to define a fold-change in gene expression, a measure of the relative change in gene expression upon the addition of inducer. One problem with measuring fluorescence in cells is that the cells are fluorescent themselves! This means that if you measure the fluorescence of a cell expressing YFP you will actually be measuring the fluorescence of the YFP molecules on top of the autofluorescence of the cell. In order to account for this you can measure the autofluorescence of a cell that lacks any YFP. We will define the fold-change in gene expression as Here, AutoFluo is the autofluorescence and Fluo([IPTG]) is the fluorescence from a strain grown in the presence of a certain concentration of IPTG. Note that Fluo[IPTG] refers to the total integrated fluorescence in a cell. This is because we are interested in the total gene expression in a cell. How will you correct for autofluorescence, given that cells have different areas (i.e., how should you calculate AutoFluo)? In order to quantify the level of gene expression you will need to write code in MatLab or other language of your choice. You will have to find the cells automatically and use the mask in order to integrate over the fluorescence of each cell. A.