Interlab Study

It is a good opportunity for our team to participate in the project which aims to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. And we are glad to cooperate with many other teams from around the world to solve the same problem in synthetic biology. This year's iGEM InterLab study is about could we use CFU/ml to replace the absorbance in fluorescence/od measurement.

To accomplish the goals, we use measure the fluorescence and absorbance with SpectraMax i3x plate reader. In additionally we need to get the competent DH5α and transforme the plasmids into cells. And this wiki will present the workflow and the results we get. We separate the wiki into 3 segments , and the first part is transformation of the plasmids into DH5α.

Transformation

According to the requirements of iGEM, we transformed all the parts (BBa_R0040, BBa_I20270, BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009) into competent DH5α. And we successfully get sufficient colonies in all the plates. Therefore we select 2 colonies in each plate to cultivate them In LB medium containing chloramphenicol for overnight with 37℃, 220rpm.

Calibration 1: OD600 Reference point - LUDOX

Through this calibration, we could transform the absorbance (Abs600 ) data from plate reader into a comparable OD 600 measurement in the spectrophotometer, which will help a lot during the following experiments. By doing so, we get the data following.

Table 1: We get the ratiometric conversion factor of 3.549 in our plate reader after measuring the wells adding 100ul Ludox and water.

Calibration 2: Particle Standard Curve - Microsphere

Because the monodisperse silica microspheres are similar to the cells in size and optical characteristics during measuring the absorbance in 600nm. It is necessary to construct the particle standard curve and in this way we could change the absorbance into estimated cells.

Table 2 :the raw data we get after diluting a series of monodisperse silica microspheres in the 96 cell well plates.　

The standard curve are straight line in both the line and log scale. So the standard curve may be credible after we use it in following experiment and the analyzing results.

Cell measurement

The fourth segment is the results of absorbance 600nm and fluorescence of transformed cells. According to the protocol we get the data and comparing them with the results in CFU/ml to get our conclusion whether CFU/ml could replace the absorbance in fluorescence/od in laboratory works.

Table 4: The raw data we get from the plate reader after measuring the samples from 0h and 6h during cultivation of the transformed cells.

After synthesis the raw data that we get from the calibration protocol and cell measurement protocol, there are also some forms after analyzing. (for easily to compare the difference between the devices, there are some diagrams only present after cultivating 6h, because we think some data getting from samples in 0h is less valuable )

Table 5: The unit scaling factors of our standard curve in calibration experiment.

Table 6: The net absorbance 600nm

Table 7: The net Fluorescence data

Table 8: The um fluorescence/OD data

Discussion: According to the net fluorescence per od value, the order of the strengths of the different promoters is estimated based on the average of two colonies in each group. The conclusion is that "device 4">"device 5">"device1" approximately equal to "positive control" >"device 2">"device 6">"device 3">"negative control". The results of the comparison with the official RFP were basically the same, while the positive and negative controls and the repetition of the two colonies indicated that the results were credible.

Table 9 : The unit scaling factors of our standard curve in calibration experiment.

Table 10: MEFL/particle

Discussion : MEFL/particle also show the same results that device 4 have the stongest promotor and device 3 have the weakest promotor. And the decreasing promotor strength is "device 4">"device 5">"device1" approximately equal to "positive control" >"device 2">"device 6">"device 3">"negative control", which corresopnd to the results in flourescence per od .

CFU/ml data in Neg.Control and Pos.Control .

We dilute the neg.control and pos.control into the absorbance into 0.1, and we finish the following dilution according to the protocol. The protocol is easy to understand and there presents the data we record during experiment.

Table 11: The original absorbance of neg.control and pos.control after dilution in plate reader

Table 12: The colony numbers in plates

we selectively choose some data to calculate the final CFU/ml . The selective standard is according to the plate colony numbers which is between 30-300. After analyzing the final CFU/ml in the negative and positive control, we found that although the origin absorance are similar, there are some difference between the CFU/ml. we are not sure whether there exists pipetting errors during dilution process . But all the data we got shows that the expressing GFP consistantly in Pos.Control may affect the growth of bacteria .

Final conclusion

We could conclude that there are some relationships between the fluorescence/OD and fluorescence per particle, So it is definitely that we could use particle of cells to replace the Absorbance of cells. However, there is no obvious relationship between the particles of cells and the CFU/ml in culture. Because CFU/ml presents the numbers of survival cells and the bacteria which have relatively strong vitality. Compared with positive control, negative control obviously have a higher chance to express antibiotic resistant gene instead of the unnecessary GFP. So when transformed cells grow on the plates, there are more chances to survival in the antibiotic plates for cells who express more less unnecessary proteins.

Of course, there may exists some pipetting errors during dilution process, but the data we obtain from the experiment show that we may can’t normalize colony-forming units (CFUs) instead of OD during fluorescence measurements .

Team:WHU-China/InterLab