From 2014.igem.org

Result

Construction of devices and sequencing

Plasmid BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector is already prepared in in official biobricks (see Fig-1), which is sequenced all correct.

Fig-1: the original plasmid of K823012-pSB1C3 to K823012-GFP-pSB1C3

BBa_J23101 + BBa_E0240 (B0032-E0040-B0015) in the pSB1C3 vector and BBa_J23115 + BBa_E0240 (B0032-E0040-B0015) in the pSB1C3 vector are constructed by us (see fig-2), which are also sequenced all correct. All of the three Plasmids are transformed into E.coli DH5α for data collecting.

<p1>(click on the picture to see more clearly)</p1> <a class="fancybox" rel="group" href=""><img src="" alt="" /></a>

Fig-2: Constructed target bricks

<p1>Left: restriction and ligation of the plasmid K823012-pSB1C3 to K823012-GFP-pSB1C3
Right: Restriction and ligation of the plasmid E2040-pSB1C3 to E2040-GFP-pSB1C3</p1>

Note: The sequences of the parts were sequenced by Guangzhou IGE Biotechnology LTD.

Data collected by microplate reader

<p.The Quantities directly gathered by microplate reader are O.D.600nm and fluorescence 480nm/515nm (main excitation peak) as

well as 480nm/540nm (secondary excitation peak) of LB culture of each strain (See Figure-3). We use RPU (Relative promoter

unit) to measure the intensity of a specific promoter (see figure-3). Besides, we abandoned RFP data because the RFP expression

of each strain with same biobrick is extremely unstable.

Fig-3: the result of promoter intensity measurement by microplate reader

<p1>The quantity of GFP expression per 1 OD directly reflects the intensity of a specific promoter. RPU shows the intensity of each promoter comparing to standard I20260.</p1>

Data processing：

The RPU is standard units proposed by Kelly J.[1], in which the transcriptional strength of a promoter can be measured using a reference standard, just like the ground in electric circuits. In the measurements of this interlab study we used a modified formula a bit different from the original one.

RPU is computed as the following formula:

in which:

&nbsp&nbsp&nbsp&nbsp->φ is the considered promoter;
&nbsp&nbsp&nbsp&nbsp->r is reference the standard BBa_I20260 and b is negative control (DH5α without plasmid in our experiments);
&nbsp&nbsp&nbsp&nbsp->dF/dt is the fluorescence of the culture, measured by microplate reader. For BBa_I20260 and DH5a, (dF/dt)/ABS is the mean of all samples; for considered promoters, (dF/dt)/ABS is the mean of the replicates of one clone. ABS is the blanked absorbance (O.D.600) of the culture, measured by microplate (not comparable with the 1 cm pathlength cuvette). Standard deviations(u) of the RPUs now can be computed as the following partial differential equations:

NOTE: We selected all the fluorescence data (unit: a.u) whose mean is within limits of ±5% compared to the relative fluorescence value.

Discussion

<p.We measure the fluorescence at different emission wavelength (515nm, main peak, and 540nm, side peak) and noticed that for

promoter (BBa_J23115 in this study) may not have obvious difference from non-fluorescence strain, especially the emission

wavelength 540nm.

Table-1: stability of GFP strains

RPUs would be different at different wavelength; the results of side peak were generally higher than those of main peak, following by bigger errors (see table 1). Thus we preferred the RPUs measured by emission wavelength 515nm.

We also noticed that, in many cases, the errors of references (BBa_I20260 and DH5a) accounted for a large proportion in the computed standard deviation (not shown), suggesting that references should be well controlled.

Flow Cytometer

Fig-4: the result of GFP measurement by flow cytometer

<p1>For each graph, X-axis is the Logarithmic distribution fluorescence intensity of each cell; Y-axis is the number of counted cells (totally 10000 counts). The data collected by flow cytometer is in accordance with microplate reader data.</p1>

Data gathered by flow cytometer shows a similar result to plate reader data, where J23101 is rather strong and J23115 is weak. GFP data is gathered (see Fig-4) but RFP is unavailable to detect because the instrument does not have a proper filter.

There is, unsurprisingly, similar results between FCM and plate reader, indicating that evident difference of promoter intensity can be observed by FCM as well, which is in tune with date from plate reader. However, here we cannot confirm whether these FCM data represents “single E. coli”, because we used none-fluorescence DH5α to adjust instrument parameter but beads of standard diameter are not used as inner reference.

All data have been submitted to official email. We are glad to participate in Interlab Study.

Reference:

[1]: Jason R Kelly et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering 2009, 3:4 doi:10.1186/1754-1611-3-4

Team:SYSU-China/file/Judging/Interlabstudy/Result.html