Team:GDSYZX/InterLab

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Interlab

Our experiment is composed of two big steps which composed of a few small experiment, here is a list of all the protocols of all those steps. The first step of the two big steps in our experiment is to make a calibration, which should be completed before the cell measurements. To do this, we should make three sets of unit calibration measurement, which including an OD600 reference point, a particle standard curve, and a fluorescein standard curve. Let’s talk about the first steps of the calibration—— reference point- LUDOX protocol. By doing this we prepared 1ml of LUDOX CL-X, ddH2O and 96 well plate. Then we add 100 μl LUDOX in to we’ll A1,B1,C1and D1. We also added 100 μl of ddH2​O​ into wells A2, B2, C2, D2. Measure absorbance at 600 nm of all samples in the measurement mode our plan to use for cell measurements is the following. Finally we record the data which showed in the table below.

(Table 1. OD600 reference point)

Table 1. OD600 reference point Then is the second steps of calibration—— Particle Standard Curve- Microsphere Protocol. In this steps of experiment we need to prepare 300 μL Silica beads - Microsphere suspension, ddH2​O, we also need 96 well plate. Then we used the method provided by the official to finish the experiment. As a result we acquire the Microsphere Stock Solution. After that, we start to prepare the serial dilution of microspheres, which is a bite of complicate. The result is showed as below.

(Picture 1. Particle Standard Curve)

Then, we begin to do the third step of Calibration—— Fluorescence standard curve-Fluorescein Protocol. In this experiment we prepare some Fluorescein, 10ml 1xPBS pH 7.4-7.6 and 96 well plate. And then we follow the official method to prepare the fluorescein stock solution as well as the serial dilution of fluorescein. Here is our Fluorescein Standard Curve.

(Picture 2. Fluorescein Standard Curve)

After the mention, we finish the first big step, now let’s go to the second big step of the experiment which is the Cell measurement Protocol. In cell measurement we should use the same plates, volumes and the same setting. These materials are needed: Competent cells (​Escherichia coli​ strain DH5α) LB (Luria Bertani) media Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH) 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) Incubator at 37°C 1.5 ml eppendorf tubes for sample storage Ice bucket with ice Micropipettes and tips 96 well plate, black with clear flat bottom preferred (provided by team) Devices (from Distribution Kit, all in pSB1C3 backbone) With those material we use the official method to start our measurement. After all we acquire the result, please check the linking below for details.

You can also download our datas

Finally we start to do the final Protocol: Colony Forming units per 0.q D600 E.coil culture. We need to do the following. Step 1:Starting Sample Preparation Step 2: Dilution Series Instructions Step 3: CFU/mL/OD Calculation Instructions. We finish all of these and get the final result and conclusion.