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Team:USTC CHINA/Modeling/B.SubtilisCulture
From 2013.igem.org
【Why Do We Design This Experiment】
Bacillus subtilis has been widely applied as engineered bacteria, especially in food industry and pharmaceutical industry, for its safety and excellent secretion capacity. Therefore, after comparing characters of distinct mutants we selected Bacillus subtilis WB800N mutant as our engineered bacteria and looked up plenty of papers to select the optimal conditions for our experiment. To our disappointment, very few experiments have been done on WB800N mutant, and most optimization experiments regarding Bacillus subtilis focus solely on the optimization of production of specific proteins produced by Bacillus subtilis. Consider the final goal of our project, it is imperative to design this experiment on our own to find out the best condition for Bacillus subtilis WB800N.
【Methodology】
Any optimization designs will inevitably involve the ideology of Design of Experiment (DOE), which includes several dependent plots. Among them Orthogonal Design and Response Surface Design, RSM for short, are the most common two in biological experiments. Generally, Orthogonal Design consumes less time and has been used more widely, yet it is not logically rigorous in mathematics, and sometimes it overlooks interactions and alias between or among factors. In contrast, RSM is constructed on rigorous mathematical theories and excels in data analysis. Having weighing the features of the two methods carefully, we finally chose RSM.【Sweeping Factors】
The first step of any methods of DOE is to investigate all variables that affect the results and select controllable factors for the experiment. In terms of this experiment, all factors can be categorized into two kinds: environment factors, like temperature, the rotation speed of the shaker, and the components of the medium. We have looked up several papers about the optimization experiments on Bacillus subtilis, finding the rotation speed of shakers ranging from 100 r/min to 250 r/min, and generally rotation speed only plays a tiny role. Additionally, our lab has only two shakers. While we can place twenty different mediums into one shaker at a time, we must run the shakers every time we alert the speed, which surely consumes longer time. Thus, we fixed the rotation speed of shakers at 200r/min.However, temperature and inoculation time are both vital environment factors whose effects cannot be ignored. Inoculation amount and pack amount are also two factors that affect results slightly. We fixed them at 5 percent and 30mL/500mL respectively according to earlier authentic experiments. A typical medium consists of carbon source, nitrogen source and inorganic salt, all of which are essential to ensure the regular metabolism of engineered bacteria. Finally in light of convenience, we infered the components of typical LB medium and determined three independent medium factors: peptone, yeast extract and sodium chloride (NaCl). Peptone provides nitrogen and carbon for the colonies, while yeast extract contains most required inorganic salt, therefore we did not list any inorganic salt except NaCl. We had no idea why NaCl is listed alone, and we suspected the influence of NaCl as yeast extract had already contains sodium. Thus, we had five independent factors: temperature, inoculation time, peptone, yeast extract and NaCl. We further investigated some papers and defined their ranges. The following table displays their levels, and the unit of peptone, yeast extract and NaCl is g/L:| Factor | Low | High |
| Temperature | 25℃ | 35℃ |
| 25℃ | 12h | 24h |
| Peptone | 5 | 15 |
| Yeast Extract | 2.5 | 7.5 |
| NaCl | 5 | 15 |
【improving】
In our experiment , we discovered that the promoter P43 is too weak to express amilCP for a blue-color display. So we redesigned the circuits , aiming to find a more feasible plan, and test the part amilCP simultaneously. The new disign contains two circuits:1 Pgrac-amilCP-Terminator
We use the Promoter grac , a promotor with lac promotor ,on the PHT vector to express amilCP, instead of Promoter 43. The Promoter grac is strong enough ,so we can easily see the results theoretically.
pgrac-amilCP overlap PCR（Right three tracks） PSBC3 Double Digestion（Left four tracks）
