Biomod/2015/Tianjin/Protocol

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  • <a href="#Preparation of cell lysate">1 Preparation of cell lysate</a>
  • <a href="#Fabrication of microfluidic device">2 Fabrication of microfluidic device</a>
  • <a href="#Preparation of microdroplets">3 Preparation of microdroplets</a>

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<a name="Preparation of cell lysate" id="Preparation of cell lysate"></a>

1. Preparation of cell lysate

The cells (E. coli strain BL21) were grown at 37 °C. When OD600 reached about 2, cell suspension was washed and centrifuged, and the resultant cells pellets were suspended and disrupted. Last, the crude lysate was centrifuged, and the supernatant was collected. The lysate used for IVTT contained 30 % E. coli cell extract, and other components including Hepes–KOH (pH 8.2), ATP, CTP, GTP, UTP, DTT, tRNA mixture, cAMP, potassium glutamate, ammonium acetate, magnesium acetate, folinic acid, 20 amino acids, creatine phosphate, and creatine kinase. The lysate mixture was divided into small aliquots and stored at −80 °C for further use.


<a name="Fabrication of microfluidic device" id="Fabrication of microfluidic device"></a>

2. Fabrication of microfluidic device

We fabricated microfluidic devices by combining photo lithography and soft lithography. A poly(dimethylsiloxane) (PDMS) replica of the channel design was formed by mixing the PDMS oligomer and curing reagent with a ratio of 10 : 1 (w/w) and curing the degassed mixture at 65 °C for 2 hours. Thereafter, access ports were bored into the soft replica with a needle. We boned PDMS replica to a glass slide after oxygen plasma treatment. The bonding process was completed in an oven at 90 °C for 1 h. Microfluidic devices were connected to syringe pumps via plastic tubing.


<a name="Preparation of microdroplets" id="Preparation of microdroplets"></a>

3. Preparation of microdroplets

The set-up includes microfluidic devices, syringe pumps, syringes and microscopy. IVTT mixture and mineral oil were hold in two syringes. We used plastic tubing to connect syringes and devices. The syringe pump was employed to control the flow rate of IVTT and oil phases. We set the flow rate ratio 1: 10 (IVTT: oil), such as 50μl/hour (IVTT): 500μl/hour (oil). At the outlet of the device, we used a vial or a home-made device to collect the microdroplets.


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