Team:ShanghaitechChina/InterLab

Abtract

InterLab is an important part in IGEM competition, whose topic is the reproducibility of protein expression(mainly GFP) from engineered biological constructs in E.coli.[1] It published an interesting but universal question， can a same device work with equal efficiency under a standard protocal and condition.

We participated in InterLab project this year. We completely finished the work of measuring the expressional level of GFP under different promotor through the method of flow cytometry. With following the standard protocal strictly, the data is reliable.

The model of flow cytometry is BD LSRFortessa X-20, collection of the data by the software BD FACSDiva 8.0.1, analysis the data by FlowJo7.6

Method

（1）Transform following 5 devices into DH5a(prepared by LiuYi) following the official standard

• Positive control
• Negative control
• Device 1: J23101+I13504
• Device 2: J23106+I13504
• Device 3: J23117+I13504

（2）Pick 3 different colonies containing the same device of all five in 5ml LB with a 10ml culture tube overnight(37°C at 220rpm)

（3）Dilute the cultures to a target OD600 of 0.02

（4）Incubate the cultures at 37°C and 220 rpm for 6 hours

（5）Dilute cells to the appropriate density about 10000 events /ml LB culture

（6）Adjust the the voltage gate of SSC and FSC to capture targeted events

（7）Adjust FITC/GFP PMT voltage(the excited light wavelength at 488nm for GFP and the sensor at 530/30 (meaning that a bandpass with 530 nm center with 30 nm width)

（8）Make 3 parallel experiment

（9）Analyse the Data

Result

click to enlarge All of the following data contain

(1) a SSC vs FSC figure to find the targeted cell.

(2)a SSC vs FITC diagram to count the strength the fluorescence.

(3) a histogram to calculate the frequency.

(4)Final value of fluorescence intensity in geometric mean.

For Negtive result

we find a low geometric mean in NC for it is set as a control

For Positive result

we find a medium geometric mean compare to NC

For Measurement Kit I

it is the strongest promotor amang 5. We can observe the green fluorescence by bare eyes.l

For Measurement Kit II

it had the a general promotor which is a little stronger than PCl

For Measurement Kit II

it had a weak promoter which is just higher than NC but we can observe it just through a snesitive set of sensor.

Reference

[1]Beal, J., Haddock-Angelli, T., Gershater, M., Mora, K. D., Lizarazo, M., & Hollenhorst, J., et al. (2016). Reproducibility of fluorescent expression from engineered biological constructs ine. coli. Plos One, 11(3).