Team:DTU-Denmark/Achievements/Interlab study

Construct Strains

We constructed multiple strains expressing GFP from different promoters of the Andersen Library. 12 out of the 15 promoters we intended to use were successfully transformed into DH5α. These 12 constructed strains were applied in the further analysis of the relatively promoter activity. The promoters J23111, J23117 J23109 were not successfully cloned into E. coli. We did not detect any fluorescence signal from these strains and our sequencing results confirmed that they were not correctly constructed. These 3 strains were excluded from the dataset. To measure the background signal a strain containing the E0240 BioBrick (GFP without any promoter) was used. A strain with the I20260 BioBrick was also applied. The I20260 BioBrick also contain the J23101 promoter upstream of GFP, but is present in another backbone than the remaining promoters.

Fluorescence Measurement on Cultures

Fluorescence was measured in the BioLector. A BioLector flower plate was inoculated with 15000 fold diluted O/N culture of the 12 above mentioned strains, the inoculations were performed in triplicates. Biomass and fluorescence were measured during growth. Fluorescence signal through the growth was normalized by dividing by OD620. The average of the fluorescence is illustrated in the bar chard below, together with the expected values relative to the J23100 promoter. Orange is the expected values and gray indicates our measured fluorescence signal. The values indicated in the bar chart are mean values of all the measurements taken during a 3-hour period, with 4.88 min intervals. For more details on the measured values and the complete dataset look at the interlab form.

The data was analyzed by fitting a statistical mixed model to the data, using the lmerTest package in R. The different promoters were modeled as a fixed effect, and replicates were modeled as a random effect. The estimated means for each promoter and the standard error of the mean can be seen in the chart below.

Figure 1 The detected fluorescence signal, with standard deviations, from 11 different Anderson promoters, presented as orange brackets. Gray brackets illustrate the expected fluorescence signal, calculated as a value relative to J23100. I20260 is also containing the J23100 promoter, but at another backbone.

In two of the triplicate sets, one outlier was excluded: For the promoter J23100, one replicate did not show significant growth. For promoter J23102 one replicate had negligible fluorescence.
The background-subtracted values were calculated by subtracting the mean of E0240 from the other means. The replicate standard deviation was estimated to be 0.014, meaning that the effect of using multiple replicates contributes a random effect distributed with this standard deviation. The residual standard deviation was estimated to be 0.040, meaning that the random effect of taking multiple measurements on the same sample has this standard deviation.
From the data we conclude that many off the evaluated promoter activities are to some extend in agreement with the expected activity. However it seems that the weak promoters are rather difficult to measure in the BioLector. We consider it an opportunity that the J23118 and J23119 at some point can have been exchange by mistake since these show reverse tendencies. However we believe that we have been careful with marking our tubes and keeping track on our samples.

This interlab study is a contribution to the bigger research collaboration. It will be interesting to see contributions from the other labs and whether these are in agreement with our obtained data.

Single Cell Fluorescence Quantification

To measure the fluorescence associated with individual cells Fluorescence-activated cell sorting (FACS) was applied on the cultures. We selected 5 promoters with significant difference in activity for measurement. Histograms of fluorescence values for each construct are shown below. At least 100,000 cells were measured for each construct. Values on the X-axes are log10-values of the fluorescence measurements.
For each construct, a threshold was determined to split the measurements into high-fluorescence and low-fluorescence cells (indicated with a red line on the figures below). The high-fluorescence sub-datasets were log10-transformed and a normal distribution was fitted. Mean and standard deviation for the log-normal distributions are reported below each histogram. The vertical red line indicates the threshold.

Figure 2: Histograms obtained from FACS measurements on strains with different plasmids containing GFP after the 4 promoters J23101, J23115, J23118, J23119 and the I20260 with J23101 followed by GFP. Mean and standard deviation is given in the histograms. The vertical red line indicates the threshold.

As we observe from these histograms the J23118 and J23119 are the strongest promoters. This is in agreement with the above described fluorescence measurement on the cell cultures. Here J23115 also induces the weakest signal.

For more detailed information on the instruments used, settings and the measured quantities see the filled out interlab form.