Team:Fudan/Measurement
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Measurement
Measurement of standard curves
No measurement is comparable if the data isn’t calibrated. calibration is always our first concern when it comes to measurement. To acquire precise standard curves, we strictly follow iGEM calibration protocols on fluorescent and Abs600. Results are shown as follows.
Figure 1~4 Particle OD600 standard curve and fluoresce standard curve captured from iGEM standard data analysis template, both shown in normal scale and log scale.
We would like to note that due to the precision limit of our 96-well plate reader, we can’t provide precise data when OD600 is below 0.002. However, we don’t believe that this would cause huge effect on our experimental data because all useful OD600 data we acquired in experiments are above 0.01. under this circumstance, distortion in standard curve doesn’t affect calibration of our experimental data.
Measurement: R0040 series
Protocols are well documented on R0040 part registry page. We also provide a link to PDF format of R0040 characterization protocol here: R0040 measurement.
As is documented in parts registry, this year our team constructed 3 parts: BBa_K3245007，BBa_K3245011 and BBa_K3245012 to characterize R0040. Therefore it’s crucial for us to maintain the repeatability of our data. To achieve this, each experiment group was repeated three times, and when sampling we put all Falcon tubes on ice to stop bacteria further growth so that the induction length was controlled to exactly one hour.
To ensure the reliability of our data, wild-type DH10B was used as the negative control.
Due to the usage of cytotoxic tetracycline as our inducer, we set a GFP constant expression system, BBa_K3245013, as the positive control to find its influence on the growth rate of Nissle 1917. Measurement is done by using a 96-well plate.
Calibration of data is produced according to the standard curve mentioned above by using ‘iGEM 2019 Data Analysis Template Fluorescence Standard Curve Protocol v2’. By conducting calibration, we eliminated potential influence of individual difference among bacteria, thus enabling our data to be more comparable.
Measurement: R0062 series
protocols are well documented on R0062 part registry page. We also provide a link to PDF format of R0040 characterization protocol here: R0062 measurement.
Since R0062 and R0040 are both regulatory promoters, we focused on acquiring more accurate data to perform better measurement. To avoid background noise from disturbing our results, we set wild type DH10B as negative control. Data are acquired and calibrated the same way as we did to R0040 data.
It is notable that we set up a new method of characterizing luxpR family promoters for future teams. We first proposed to use OD-MEFL/particle to characterize luxpR and its analogies.
We discovered that, the more plasmid transformed into bacteria, the slower it grows. Under this circumstance, incubation time no longer stands completely for growing stage or concentration of bacteria, but we can always use OD600 to precisely represent bacteria concentration. So we drew OD600-MEFL/particle curve and conducted nonlinear regression on it.
Of the four fitted value, what we care about most is the exponent that is above 1. In this case it’s B. This B stands for robustness of this luxpR promoter. The higher exponent value a luxpR promoter shows, the stronger ‘switch character’it has. In our project luxpR-HS100(BBa_K3245009) reached over 2 in B value, which means that it has strong robustness and that our modification was successful in an unexpected aspect——improving the robustness of a regulatory promoter. Teams in the future that wants to characterize luxpR family promoters can all benefit from this new analyzing method that we put forward.