Contents

0. Summary

1. ssrA tag

2. RNA thermometer

2.1.“Background”for our characterization

2.2. Summary of the experiment

2.3. Result

2.3.1 The fluorescence intensities of RFP

2.3.2 The standardized fluorescence intensities of RFP after subtracting the background derived from the Negative control cell

2.4. Discussion

0. Summary

iGEM 2015 Tokyo_Tech improved, by model-based approach,the function of BBa_K395160 by submission of BBa_K1632020 and BBa_K1632023, and characterization of BBa_K1333309, to meet gold medal criteria.

Our project requires improvement of a previously existing part for Chloramphenicol resistance (CmR) coding sequence (BBa_K395160 iGEM 2010 Tokyo_Tech) to decrease unintended antibiotic effect by leaky expression, because we had to diminish such effect for implementation of a payoff matrix in Prisoners’ Dilemma. Since our modeling suggested that such improvement is most efficiently achieved by increased degradation of the resistant protein, we designed CmRssrA (BBa_K1632020), and fused it to an inducible promoter to obtain BBa_K1632023(Fig. 7-1-0-1A). In the improved part, ssrA tag fused to CmR coding sequence with successfully degrade the leaked CmR protein, solving the problem of leaky expression of CmR in our wet experiment (Fig. 7-1-0-1B) (1. ssrA tag). We have also submitted parts for silver and bronze medal, respectively.

Fig. 7-1-0-1.

Furthermore, we have improved characterization of another previously existing part, RNA thermometer (FourU) coding sequence plasmid (BBa_K1333309), constructed by iGEM 2014 SYSU-China by the following three points. (1) We measured the function of BBa_K1333309 at 42ºC, which wasn’t confirmed by iGEM 2014 SYSU-China. (2) We clarified the processing of the background derived from Negative control. (3) We explicated that the measuring equipment was flow cytometer. (2. RNA thermometer)

1. ssrA tag

Although growth inhibition by chloramphenicol is required for implementation of our payoff matrix (Fig.7-1-1-2A), cells showed active growth even in the absence of AHL when the cell harboring our initially designed genetic circuit Pcon_rhlR_TT_Plux_CmR in Prisoner A coli (Fig.7-1-1-2B).
	Fig. 7-1-1-1. Our initially design of a part containing a previously existing part

To solve this problem, our series of modeling suggested us select next circuits designs, one of which is addition of ssrA degradation tag to the C-terminal of the chloramphenicol resistant protein (CmR). Firstly, we adjusted the model to include leaky CmR protein expression found in previously existing part (Fig. 7-1-1-2 C) (Further information of modeling is in here). We then planned two solutions, each of which is evaluated by modeling, to circumvent the unintended antibiotics resistance due to leaky expressions (Fig. 7-1-1-2 D and E.). Because increase of degradation rate of CmR showed more effective suppression of growth (Fig. 7-1-1-2 D) than increase of Cm concentration (Fig. 7-1-1-2 E), we created CmR coding sequence with ssrA degradation tag (BBa_K1632020).

Fig. 7-1-1-2. We compared the results in modeling and wet lab

In E. coli cell culture, as predicted by the modeling, the ssrA tag suppressed unintended growth by degradation of the leaked chloramphenicol resistant protein (CmR). The improved part BBa_K1632020) was used for construction of BBa_K1632023 circuit for C4HSL inducible expression of CmR. Compared with circuits without ssrA tag BBa_K1632025 (Fig.7-1-1-3 green dotted line), our improved BBa_K1632023 indeed showed much slower growth (Fig. 7-1-1-3 pink dotted line). Furthermore, addition of C4HSL recovers active cell growth to show antibiotic effect from CmRssrA (Fig. 7-1-1-3 pink solid line). These results show the improved function of AHL-dependent CmR expression by measuring the concentration of cells. Please see here for further information.

Fig. 7-1-1-3.

2. RNA thermometer

2.1. "Background”for our improved characterization

Temperature increase is required for an RNA thermometer in the enhanced expression of BBa_K1333309 (J23119_K115002_E1010) constructed by iGEM 2014 SYSU-China. The RNA thermometers are located in the 5’-untranslated region (5’-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing. Translation initiating temperature allows the disconnection of the coupling of the hydrogen bonds, which block the SD sequence at low temperature. Therefore, RNA thermometers change their conformations to the open state so that the ribosome could access the SD sequence and to initiate translation.

Although iGEM 2014 SYSU-China reported larger temperature dependency of RFP production dependent on the RNA thermometers than the dependency of production without the thermometer, it is hard to evaluate their results due to the lack of descriptions about negative controls. Their graph indeed reported fluorescence intensities at 30ºC and at 37ºC for RFP expression controlled by the RNA thermometer. They further calculated the ratio in the above two expressions. Additionally, intensities and ratio were reported also for RFP expression not controlled by the RNA thermometer. They, however, have not reported how theseintensities were acquired or ratios were calculated. Furthermore, they have reported neither the fluorescence intensities nor the increasing ratios of the fluorescence intensity, of the sample [NC] at 30 ºC and at 37 ºC. Background fluorescence of negative control should be considered for such ratio calculation, especially in case of this part with low fluorescence intensity, which is shown afterwards in our Discussion 3-3-3. Given this situation, it can be speculated that the fluorescence intensities of the samples at 30 ºC and at 37 ºC written in the graph, are the values of each measured fluorescence intensity subtracted by the fluorescence intensity of Negative control. However, evaluation for interchangeable parts requires precise protocols they have not disclosed. A minor issue in their protocol is the lack of information on whether the measurement was done with the plate reader, which is affected by the medium and the fungus density, or done with the flow cytometer, which is not affected by these factors.

We thus improved characterization of BBa_K1333309 by (1) measuring the function of the part at 42ºC, (2) explicating the way to deal with the background derived from Negative control, (3) measuring with the flow cytometer.

2.2. Summary of the experiment

Our purpose is to confirm the behavior of the RNA thermometer by setting Positive control and Negative control and to characterize the temperature dependency of the RNA thermometer at 30ºC, 37ºC and 42ºC by using the flow cytometer. We prepared the samples as shown below.

BBa_K1333309: J23119 promoter_RNA thermometer_rfp (pSB1C3)
Positive control: Plac _rfp_TT (pSB1C3)
Negative control: RNA thermometer_rfp (pSB1C3) (Promoter-less control)

2.3. Results

We measured each sample at 30ºC, 37ºC and 42ºC. The translation initiating temperature is 37ºC. Little background from medium affect results for flow cytometer. Although iGEM 2014 SYSU-China confirmed the function of Pcon_RNA thermometer_rfp at these temperatures, we additionally measured each sample at 42ºC, which is higher than the translation initiating temperature.

2.3.1 The fluorescence intensities of RFP

We found that the fluorescence intensities of both Pcon_RNA thermometer_rfp and Plac_rfp increased along with the rise of the temperature (Fig. 7-1-2-1).

Fig. 7-1-2-1. RAW data

The error bar represents the standard deviation of two samples which derived from two different colonies, respectively.

2.3.2. The standardized fluorescence intensities of RFP

2.3.2.1. The standardized fluorescence intensities of RFP

  We obtained increasing ratios of fluorescence intensities at 37ºC and at 42ºC by dividing the each of the raw fluorescence intensities (Fig. 7-1-2-1) by those at 30ºC. The increasing ratios of the Plac_rfp at 37ºC and 42ºC show that the fluorescence intensities, even without the RNA thermometer, increased dependent on temperature. We further evaluated the increasing ratios of Pcon_RNA thermometer_rfp at 37ºC and 42ºC. Compared to the increasing ratios of Plac_rfp, those of the Pcon_RNA thermometer_rfp were higher at respective temperatures. This comparison shows not only the increase in the fluorescence intensities dependent on temperature, (Fig. 7-1-2-1) but also the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 7-1-2-1). The increasing ratio of Pcon_RNA thermometer_rfp was 1.3 times higher than that of Plac_rfp at 37ºC. Furthermore, the increasing ratio of Pcon_RNA thermometer_rfp was 3.2 times higher than that of Plac_rfp at 42ºC. These differences of the increasing ratios were dependent on the function of the RNA thermometer. We concluded that the RNA thermometer shows higher function at 42ºC compared to 37ºC.

Table. 7-1-2-1. The increasing ratios

2.3.2.2. The standardized fluorescence intensities of RFP after subtracting the background derived from the Negative control cell

  In this section, we obtained processed fluorescence intensities by subtracting the fluorescence intensity of a negative control, the RNA thermometer_rfp (Fig. 7-1-2-1) from both the fluorescence intensities of Plac_rfp and Pcon_RNA thermometer_rfp (Fig. 7-1-2-1), each at the same temperature. We then obtained increasing ratios with background subtraction at 37ºC and at 42ºC by dividing the each of the processed fluorescence intensities by those at 30ºC (Table. 7-1-2-2). The increasing ratios of the Plac_rfp with background subtraction at 37ºC and at 42ºC show that the fluorescence intensities increased dependently on temperature. We evaluated the increasing ratios of Pcon_RNA thermometer_rfp with background subtraction at 37ºC and at 42ºC. Compared to the increasing ratios of Plac_rfp with background subtraction, those of the Pcon_RNA thermometer_rfp were higher at respective temperatures. We observed again the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 7-1-2-2). The increasing ratio of Pcon_RNA thermometer_rfp at 37ºC with background subtraction was 2.6 times higher than that of Plac_rfp at 37ºC. Furthermore, the increasing ratio of Pcon_RNA thermometer_rfp with background subtraction was 6.8 times higher than that of Plac_rfp at 42ºC. These differences in the increasing ratios were dependent on the function of the RNA thermometer. We concluded that the RNA thermometer shows higher function at 42ºC compared to 37ºC.

Table. 7-1-2-2.The increasing ratios with background subtraction

2.4. Discussion

We examined that the reason the function of the RNA thermometer was worse at 37ºC than at 42ºC was that the amount of hydrogen bonds forming RNA thermometer was not enough to change in the structure of the RNA thermometer at 37ºC.

Furthermore, differences between the increasing ratios with and without background subtraction disclose the importance of background treatment. At 30 ºC, Pcon_RNA thermometer_rfp wasn’t translated enough and showed little expression of RFP. Since the fluorescence intensity of Pcon_RNA thermometer_rfp at 30ºC was small, the increasing ratios of the Pcon_RNA thermometer with and without background subtraction greatly differ in both 37ºC and 42ºC (Table. 7-1-2-3). Therefore, it is important to clarify whether the background derived from Negative control was processed or not.

Finally, we would confess that our measurement of the temperature dependencey of RPF expression controlled by the RNA thermometer was part of our dilemma project to express the feeling of the E. coli which fell into a dilemma.

Table. 7-1-2-3. The difference the increasing ratios with and without background subtraction