Device

Part:BBa_K4206001:Experience

Designed by: Kazuma Tsurumaki   Group: iGEM22_Qdai   (2022-10-02)
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Applications of BBa_K4206001

Experiment
1. Gibson assembly 

   Forming the plasmid contains aatA and poxB.

2. Transformation 

   Following the Single Tube Transformation Protocol provided by iGEM, except for the process of heat shock. 
   We conducted heat-shock for 30sec at 42°C on a heat block.

3. Culturing 

   Pick colonies and culture them in LB-cam containing ampicillin overnight.

4. IPTG induction 

   Add 30μl of culture fluid to 3 ml of LB-medium containing ampicillin and 0-1.5μmol of IPTG. 
　 Cultured for 4-5h at 37℃ in culture tubes in a shaking water bath at 168 min^-1

5. Measure the cocentration of acetic acid every hour 

   * According to the protocol offered by assay kit's manifacture, make the standard curve. 
     Since LB medium might contains acetate and absorbrance, acetate samples used in calibration was made by dliting acetate 
     Standard with LB mediums instead of water. 
   The protocols is here; https://www.bioassaysys.com/datasheet/EOAC.pdf
   *  Sampleing  culture medium and mix with the working reagent of assay kit.
   * Mesure absorbrance of 455-630nm by spectrometer (USIO Picoscpe) every hour to determine the concentration of acetic acid　
     based on the standard curve.
   * OD600 was measured too by spectometer.

Result

As shown in the chart1, acetic acid was synthesized when 1.5 mM IPTG was added.

In the chart2, OD600 has a higher growth rate at higher IPTG concentrations. The presence of acetic acid may have a positive effect on the growth rate of E. coli. Millard et al. (2022) have argued that acetate acts as a co-substrate of glycolytic nutrients and boosts E. coli growth at low glycolytic fluxes. The change in OD600 with the addition of IPTG may be due to increased intracellular acetate concentration and growth promotion by the expression of poxB. we have confirmed the production of acetic acid, we need to use these data to explore the optimal promoter strength of poxB.

chart1
chart2






References
Millard, P., Uttenweiler-Joseph, S., & Enjalbert, B. (2022). From toxic waste to beneficial nutrient: acetate boosts Escherichia coli growth at low glycolytic flux. BioRxiv. https://doi.org/10.1101/2022.09.20.506926


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