Team:Lethbridge HS/Model





 Ink Production

      

The modeling portion of our project will allow us to estimate our pigment yields. The main pigment example that we are using is melanin. When designing the melA expression construct, we referenced from the Lagunas-Munoz et al., 2016 and we learned that the yields of melanin in E. coli are the highest at a temperature of 30 degrees Celsius and at a pH of 7.0. We will be using their experimental results as a model for what we should be expecting during our attempts to produce the melA tyrosinase and melanin in E. coli. As our system is modified from the system used in this paper to account for BioBrick standards and what we have available to us in the lab, we may be able to explain some differences in our production levels. We are using a different plasmid, cell strain, and will initially be using richer media.

The graphs below summarize some of the data obtained by Lagunas-Munoz et al., 2016. Firstly, they found that the cultures would reach stationary phase before melanin production would begin (or at least be detectable). Secondly, melanin would only be produced when the glucose in the media was consumed, which correlates with the culture reaching stationary phase. The group also found that melanin production was not favourable at growth temperatures of 35 degrees Celsius or higher. They found that the optimal balance between cell growth and melanin production occurred at 30 degrees. Using this information, we decided to grow our melA expressing cultures at 37 degrees until we induced expression with IPTG, then continue to grow the cultures at 30 degrees.

The graphs below summarize some of the data obtained by Lagunas-Munoz et al., 2016. Firstly, they found that the cultures would reach stationary phase before melanin production would begin (or at least be detectable). Secondly, melanin would only be produced when the glucose in the media was consumed, which correlates with the culture reaching stationary phase. The group also found that melanin production was not favourable at growth temperatures of 35 degrees Celsius or higher. They found that the optimal balance between cell growth and melanin production occurred at 30 degrees. Using this information, we decided to grow our melA expressing cultures at 37 degrees until we induced expression with IPTG, then continue to grow the cultures at 30 degrees.




The graphs below show the highest results from the Lagunas-Munoz and colleagues.

This is a graph of the data of biomass (gDWCl¯¹) vs. time (hr) at a temperature of 30 degrees Celcius.


This is a graph of the data of glucose levels (g l¯¹) vs. time (hr) at a temperature of 30 degrees Celcius.

This is a graph of the data of eumelanin (g l¯¹) vs. time (hr) at a temperature of 30 degrees Celcius.


This is a graph of the data of eumelanin (g l¯¹) vs. time (hr) at a temperature of 30 degrees Celcius.

Reference: Lagunas-Munoz, V.H., N. Cabrera-Valladares, F. Bolivar, G. Gosset, and A. Martinez. Optimum melanin production using recombinant Escherichia coli. Journal of Applied Microbiology, 2006. 101: 1002-1008.

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