Difference between revisions of "Team:NKU China/InterLab"

Good bricks make strong buildings. The same is true in the field of synthetic biology. To ensure the biobricks created in iGEM competitions are repeatable and reliable ,a robust measurement procedures been developed for green fluorescent protein (GFP) over the last three years by the Measurement Committee, through the InterLab study.Also,numbers of teams participate in this standardized measurement project.We’re honored to participate in the fourth International InterLab Measurement Study.

By providing a protocol in details as well as forms helping unify the analyse of data,the Measurement Committee hopes to handle the problems emerged while establishing a baseline for replicability of fluorescence measurements.So with the same devices,the same protocol and devices,we try to measure the fluorescence intensity controlled by this year’s devices and study the distribution pattern of those results with teams all over the world.

The following devices (from InterLab Measurement Kit) were transformed into Escherichia coli DH5α:

2 colonies from each of plate were picked and inoculated on 5-10 mL LB medium + Chloramphenicol.
Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.

200 µL of each of the 16 overnight cell cultures was mixed with 400 µL 50% glycerol and the mixture was preserved at -20℃.

Prepare the fluorescein stock solution:

Prepare the serial dilutions of fluorescein:

TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.

5µL of preservation mixture of each of the 16 colonies was inoculated in 5 mL LB medium (with 35 ng/µL chloramphenicol) in a 15 mL tube.Cells were incubated overnight for 15 h at 37℃ and 220 rpm.

Quantification and standardization play important roles in synthetic biology.Although the parameter values are calculated by model equations, it is hard to select the biobricks that reliably implements a desired cellular function with quantitative values. To overcome this problem, the RBSs were designed to control the expression of downstream genes when necessary.[1]This year we are about to test RBS B0034 and another modified RBS called bicistronic device (BCD2)-J364100 with the same GFP gene:GFP E0040.

We hope that our experimental results are direct and easy to understand,so we just let E.coli themselves tell the story by making them grow into the shape of our team name.In this picture,N is written with negative control,K&U with positive control.The last six letters were written with E.coli containing devices 1-6.It’s quite interesting that we can see device 2 is the brightest,even brighter than positive control.Among device 4-6,4 is the brightest,which corresponds with our experimental data beleow well.

Fig3. Team name written with cultures in Interlab experiment.Letter N is written with group Negative Control,K and U with Positive Control,”-”with Device 1,C with Device 2,h with Device 3 and i,n,a with Device 4,5,6.There are obvious differences in fluorescence intensity they expressed.

Analysis of Variance Table

Response: value
              Df     Sum Sq    Mean Sq  F value    Pr(>F)    
promoter       2 1.5095e+12 7.5475e+11 428.6242 < 2.2e-16 ***
RBS            1 5.9784e+10 5.9784e+10  33.9512 2.577e-08 ***
Time           3 1.6484e+10 5.4947e+09   3.1204   0.02736 *  
colony         1 2.4858e+09 2.4858e+09   1.4117   0.23636    
replicate      3 1.9372e+09 6.4574e+08   0.3667   0.77711    
promoter:RBS   2 2.7491e+12 1.3746e+12 780.6174 < 2.2e-16 ***
Residuals    179 3.1520e+11 1.7609e+09                       
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Since there are several factors like different promoters,RBSs,and interactions of the two elements can infecting the expression of GFP while time also influence the result to some extent as difference appears in the weight of influence caused by promoters and RBSs as time went by.To get more useful information,we conduct a variance analysis to evaluate the effect of these factors.Sheets above are the results we get.
Basically we have a closer look at these data by dividing them by hour and seeing how different growth status can influence the results.And as we can see in all the sheets, in general the main factors with significant difference are promoters,interaction effects and replicates,which causes us great interest.
It’s obviously that at 0 h all cultures’ conditions are almost the same.From sheets describing the data of 2 h, we can see that there are significant difference among different promoters as well as interaction effects between promoters and RBSs(elements) while different elements didn’t show that significance.Analysing the results of 4 h, we can see that promoters and interaction effects are still the factors with significant difference,while the p-value of elements(RBSs) is much smaller than 2 h.It seems that the elements function later than promoters, however, more supporting information is needed.An interesting conclusion from 6 h’s result is the contribution of elements for significant difference are larger, which seems confirmed the suppose above.That’s a quite charming discovery, maybe this can help studying the temporal regulation of gene expression regulation.

All the forms we get from the analysis show that different promoters and different interaction effects between promoters and elements have significant difference.Also we find the tendency that as time goes by, different elements shows larger and larger difference.However, all these finding and problems are open questions and we welcome any innovation helps explaining all these findings and solving problems.We can be reached at nkuigem2017@163.com.

The promoters in the devices we use this year is the same as last year's material. Generally the relative transcription initation strength of three promoters is consistent with the result characterized by RFP FI．
The results we get shows that under the standarlized protocol , different devices behaved differently and more functions can be inferred from special phenomenons.　
Firstly we felt it was harder to transform plasmid with device 1 and 6,which was confirmed by JLU.NPU also reported difficulty on device 4.
Then the index to measure-FI shows that positive control is not always the highest.This time device 2 made it.
Finally about describing the results,we think it would be better if the Fluorescein Standard Curve and microbes with GFP are in the same buffer like PBS to make errors from backgrounds least.It's also a good idea to give the equation turning FI of GFPs into their concentration.