Team:XJTLU-CHINA/Interlab

Interlab

Standardizing the measurements of fluorescence is a meaningful in scientific comparasion of data. We participated in the iGEM Interlab study following the instructions and provided by iGEM headquarter. Based on the constructs description, we made a hypothesis of the result of experiment. However, experiment results cannot support the hypothesis, so further operation have been done to make sure the correctness of the manipulation of experiment.

Parts

Three interlab devices and two control group have been study. These include:

Test Device 1: promoter J23101. RBS B0034. GFP E0040.Transcription Terminator B0015 Test Device 2: promoter J23106. RBS B0034. GFP E0040.Transcription Terminator B0015 Test Device 3: promoter J23117. RBS B0034. GFP E0040. Transcription Terminator B0015 Positive Control Device: promoter I20271, with GFP Negative Control Device: Promoter R0040, without GFP

All these devices are embedded in plasmid pSB1C3. The plasmid DNA are stored in InterLabMeasurementKit with concentration 100 pg/uL in a total of 10uL of Buffer EB. Other important materials related to Interlabstudy including the FITC Standard and LUDOX. What should be mentioned here is the LUDOX in Our own stock has been made precipated due to improper storage condition, following the suggestion from Traci@igem.org Thanks to Northeastern University of China, we get another tube of LUDOX.

Procedure

The method of measurements of fluorescence we choose is plate reader. The strain we used for transformation is Escherichia coli Top10. We obeyed the protocol provided by IGEM headquarter to carry out the experiment. Mainly, the whole experiment can be divided into the following parts, Transformation and incubation, relative fluorescence measurement.

Transformation and incubation: All five devices were transformed into E. coli top 10 which is purched from Tiangen. The heat shock time is one minutes, recover time is five minutes. Later these cells were incubated in agar plate and LB broth with chloramphenicol antibiotics.

Relative fluorescence measurement: relative fluorescence measurement includes OD measurement and fluorescence measurement. For characterizing the measurement result, OD600 Reference Point and Fluoresence standard curve have been obtained previously. In the formal experiment, 5 devices with replication and blank were pipetted into 96-well plates to obtained OD600 and fluorescence data from plate reader. However, we only have transparent and black 96-well plates, therefore we doubled everything from the protocol in this experiment: we used 20ml medium and take 200uL cultures every time - 100ul for transparent plate to measure OD600 and 100ul for black plate to measure fluorescence. In addition, our incubator can only reach maximum 190rpm so we use 190rpm to incubate the cultures.

Results

Figure.1 OD600 measurement of devices 1 to 3, as well positive control and negative control. Each group have two repetitions.

Figure.2 Absolute fluorescence measurement of devices 1 to 3, as well positive control and negative control. Each group have two repetitions.

Figure.3 Relative fluorescence measurement, plotted base on OD600 measurement and Absolute fluorescence measurement

Discussion

OD600 measurement displays the characteristic of cell growth, which shows exponential growth from 1 hour to 4 hour. No atypical points appear as well.

Absolute fluorescence also exhibits the same motif as OD600 as the fluorescence intensity grows exponentially during 1 hour to 4 hour. However, the fluorescence intensity decreases from the fifth hour. Two message from this picture should be attention. First, there is an atypical data point at the fifth hour of device 2 replicate 2 which may due to operation mistake or mechanical error. Second, except the atypical point, both repetition of device 2 show a higher fluorescence density than the device 1 throughout the time scale revealed by the plot. These phenomena will be discussed later.

In relative fluorescence measurement, device 2 shows a higher relative fluorescence than device 1 as the. Device 3 has the lowest relative fluorescence among the three devices. These characteristics can also be observed at the plot of absolute fluorescence.

Before starting the exam, we have made a hypothesis of the difference of fluorescence intensity of three devices from documented promoter strength. Based on the promoter stringency, we think that devices one, which embedded the strongest promoter should have the highest absolute fluorescence. Device 3, which promoter is the weakest among the three devices, should have lowest absolute fluorescence. Though indeed the device 3 exhibited the lowest absolute fluorescence, absolute fluorescence of device 2 is higher than device 1. First we thought that we might got device 1 and 2 mixed up however we sequenced the strains we used and made sure that we have correctly transformed those cultures. Therefore, in our experiment the devices two did exhibited higher absolute and relative fluorescence than device 1. Though this result may be minority even the only case among all the team which participate the Interlab study, we insist the accuracy of our experiment, and we also curiosity about the explanation of this result.

click here to see the interlab form and data for plate reader.