Team:UCL/Interlabstudy

'

Interlab
Study

▼

Introduction

This year's iGEM team from UCL is the first from our university to take part in the iGEM InterLab Study. The aim of the Interlabstudy is to have many labs across the world perform the same experiment and to demonstrate how results differ depending on the methods available and used in different labs. The Interlabstudy in the iGEM competition is the first of its kind at this scale and the results might be useful to improve the robustness of experimental data in the future. We found the idea of this project exciting which is why we decided to contribute to it.

The chart shows the results of our measurements on the three interlab study devices and a comparison to positive and negative controls and a blank measurement using only TE buffer (10mM Tris-HCl, 1mM EDTA). We took three clones from each device (biological replicates) and as part of the extra credit for the InterLab Study we took three measurements of each of these (technical replicates).

Summary

We investigated the strength of three different promoters that were defined by iGEM by measuring the fluorescence of a specified downstream GFP construct. We found that in this experiment the promoters differed significantly in strength, with the promoter J23101 inducing GFP fluorescence almost twice as strong as the promoter J23106. The promoter J23117 showed only slightly higher expression of GFP than the negative control.

Our Lab Procedures

Devices we measured

  1. InterlabStudy devices
    • Device 1: J23101 + I13504
    • Device 2: J23106 + I13504
    • Device 3: J23117 + I13504
      • (Click here for more information about this promoter family)
  2. Positive control
    constitutive GFP expression device: BBa_I20270
  3. Negative Control
    As negative controls we used the above promoters without downstream GFP.
    • Negative Control 1: J23101
    • Negative Control 2: J23106
    • Negative Control 3: J23117

Cloning

We used 2A assembly (standard biobrick assembly) to clone the GFP gene downstream of the promoters. We digested the backbone that included the promoter with SpeI and PstI and the GFP construct with XbaI and PstI. We then performed a ligation to assemble the parts using T4 ligase. We performed diagnostic digestions to test if the ligation step worked and sequenced the plasmids for final confirmation that the ligation.

In the final step we transformed the ligated plasmids into competent DH5 alpha cells.


Growing

Three biological replicates of each construct were used to make overnight cultures. As a positive control we used the biobrick I20270 which has a GFP gene downstream of a constitutive promoter. As negative control we used the promoters with no GFP insert. We transformed both controls from the distribution kit into competent DH5alpha cells.

From glycerol stocks, we inoculated each construct into 10ml LB cultures with chloramphenicol and incubated these overnight at 37 degrees, shaking at 300rpm.


OD measurements

The microplate reader used was a BMG fluostar optima microplate reader, set to absorbance measurement. To find the OD of the overnight cultures, we took 200ul of each overnight culture and pipetted them on to a clear 96 well plate. Then we performed two 10-fold serial dilutions with LB on the plate. We measured the OD600 of the cultures on the plate, shaking for 30s before measuring.

After measurement we calculated the volume of each culture that would give an OD of 0.5 when diluted into 1ml. We centrifuged the samples at 4 C at 12000rpm for 2 minutes and removed the supernatants. Then we resuspended the pellets in 1ml of TE buffer (10mM Tris-HCl, 1mM EDTA). Afterwards, we measured the OD again with TE buffer as blank.

Fluorescence Measurements

The microplate reader was the same as above (BMG fluostar optima microplate reader). A black 96-well plate was used. We measured the fluorescence intensity of each sample in triplicates. The excitation wavelength was ... and the reading wavelength ...

Results

The chart presents the final results for the interlabstudy promoters. The error bars represent the standard deviation from the mean.


The raw data was taken in biological and technical replicates and is shown in the chart above. The negative control represents the means of the negative control for each promoter.



Significance of the third promoter

We noticed that the fluorescence results of promoter J23117 and the negative control were very close. Hence, we performed an analysis using an anova1 test in Matlab using measurement results from nine replicates of the negative control and nine replicates of the promoter device. The plot above shows the results, indicating that the means of the samples are far apart (circles). The bars next to the means show the confidence interval of the samples which is 95%. The confidence intervals do not overlap so the samples can be interpreted as significantly different.

The p-value was also calculated at 0.001% confirming the interpretation that the fluorescence is definitely significant compared to the negative control.

Discussion