BME100 Lab 2_nigel

BME100 Lab 2_nigel - BME 100 Laboratory 2 Population...

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BME 100 Laboratory 2 Population control gene circuit Nigel Chou TA: Brian Diekman November 28, 2007 Instructor: Fan Yuan
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BME 100 Lab 1 1. Introduction Synthetic gene circuits are a part of the relatively new field of synthetic biology and show much potential the fields of medicine, green chemistry and renewable energy due to the ability to manipulate the growth behaviour of populations of cells, especially bacteria [1]. In this laboratory we will analyse the growth curve of E-coli bacterial cultures with and without a synthetic gene circuit that allows it to regulate its own population density by simultaneously broadcasting and detecting its cell density using a quorum sensing system [2]. We also examine the growth behaviour of bacteria with the gene circuit inactivated by glucose and compare it to cultures with an activated gene circuit. In this experiment, light absorbance of the media is used to quantify cell density. As a cell population increases, there is a corresponding increase in light absorbance (which leads to a cloudy appearance or ‘turbidity’) due to the presence of more cells which absorb and scatter light. Thus, by measuring light absorbance of the culture at regular intervals, the cell-density of the culture can be tracked. The absorbance is measured by the Victor3 plate reader, and the results obtained are plotted to observe absorbance trends, which are then compared between the different cultures. 2. Materials and Methods Materials 1) Wallac 1420 Victor3™ plate reader 2) Growth media for cultured strain of E-coli 3) Culture of E-coli cells without population gene circuit 4) Culture of E-coli cells with population gene circuit 5) Glucose solution 6) 48-well plate 7) 1.5ml Eppendorf tubes 8) 20 μl micropipette 9) mineral oil Methods Sterile technique is used throughout the experiment. 500μl of growth media is first added to each well in alternate columns of wells in the 48 well plate. Five eppendorf tubes are then prepared, labeled A through F. 5μl of E. coli culture without the population gene circuit is transferred into the eppendorf tube 1
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BME 100 Lab 1 labeled B. 5μl of E. coli culture with the population gene circuit is transferred into each of the eppendorf tubes labeled C, D, E and F. 12μl of glucose solution is then pipetted into each of the eppendorf tubes labeled D and F. Half of the lab groups will prepare another eppendorf tube labeled A, to which 12μl of glucose solution is added. The solutions in each of the eppendorf tubes were pipetted into their respective wells in the 48-well plate, as summarized in figure 1. Figure 2: diagram of content of wells in 48 well plate. Another set of the same configuration is done by other lab groups in two other columns 250μl of mineral oil is then added to each of the wells to prevent evaporation over the relatively long duration of the experiment. The 48-well tissue culture plate was then placed into the Victor3 plate reader, which is maintained at 37°C. Over the next 63 hours, at 30 minute intervals the samples were
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BME100 Lab 2_nigel - BME 100 Laboratory 2 Population...

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