Team:HKUST/Results

ShellBi

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Results

Results and Discussion

Overview

The processed data results are presented in this page along with discussion which helps us to proceed to the next stage of the engineering design process as seen in the Engineering page. The experiments associated with these results are described in detail under Experiments.
Our Results and Discussion involves 5 major points:

• ❒ Extracellular vs. Intracellular osmolarity
• ❒ Fluorescence Measurement of TCS Basal
• ❒ Fluorescent Measurement of mRFP1 + PompC_mRFP1 Antisense
• ❒ Fluorescent Measure of eGFP + PompF_ eGFP Antisense
• ❒ Characterization of Negative Regulator Region of PompF

Our 5 Major Experiment Results

Extracellular vs. Intracellular Osmolarity To improve the accuracy of data, we calibrated the osmometer within the scale of intracellular (0 - 50 mOsm/kg with 10 mOsm/kg increment) as well as extracellular osmolarity (100 - 1500 mOsm/kg with 200 mOsm/kg increment) before measurement. With our standard solution of NaCl, we took triplicate measurements to measure its precision (with an average S.D. of 1.58 mOsm/kg). Overall, it shows a negative systematic error.

We plotted a calibration curve to correct the measured value to its true value to obtain the final calculation with the following formula:

Measured Osmolarity = 0.839±0.004 Corrected Osmolarity - 10±3 R ²=0.9998

Osmometer Calibration Curves

Figure 1a: Osmometer calibration curve (100-1500 mOsm/kg)

Figure 1b: Osmometer calibration curve (0-50 mOsm/kg)

Figure 2: Osmometer data- Intracellular osmolarity v/s Extracellular osmolarity (100-1500 mOsm/kg)

An exponential relationship is observed between the extracellular and intracellular osmolarity. With an R2 value of 0.9629 we could reliably utilise this data for mapping extracellular medium concentration to the corresponding intracellular osmolarity. We collected data from 15 different sodium concentrations in LB. Note that the machine cannot accurately determine the osmolarity of samples lower than 25 mOsm/Kg which is why the data collected from those samples has a high standard deviation.

The subsequent readings were collected by growing cells in varying concentrations of NaCl in LB medium but the data is interpreted by mapping the external medium molar concentration to the intracellular osmolarity using this graph.

Fluorescence Measurement of TCS Basal TCS basal is an integral part of our project. This is the basal circuit which contains eGFP driven by an OmpC promoter, and an mRFP1 driven by an OmpF promoter. The fluorescence measurement as collected by the FlexStation Multimode Plate Reader was divided by the OD600 in order to normalize the intensity to cell number.

Figure 3: TCS basal eGFP fluorescence with increasing intracellular osmolarity

Figure 4: TCS basal mRFP1 fluorescence intensity v/s intracellular osmolarity

As seen from the graphs (n=4), the expression of eGFP, observed as its intensity, is increasing when the intracellular osmolarity increases. This is in line with our hypothesis as the eGFP gene is expressed under the OmpC promoter. Since this promoter is preferentially expressed under higher osmolarity conditions, the eGFP expression increases with an increase in intracellular osmolarity.

In Graph 2, the mRFP1 intensity seems to be decreasing when intracellular osmolarity increases. This result supports our hypothesis since mRFP1 is controlled by the OmpF promoter. This promoter is active under low osmolarity within a cell. We observe a massive decrease from intracellular osmolarity change from 28 to 50 mOsm/kg.

Assuming our Dry lab modelling is correct, this steep decline is due to the narrow range of functionality of OmpF promoter. Perhaps in order to characterise it completely, we would have to experiment with an even lower concentration. However, lower concentrations were found to be lethal to the cells. Any data obtained from those cells was unreliable since most cells were dead in the sample. Additionally, data from osmometry shows that the machine is not sensitive to ranges lower than 0.04M of solute and results are not reproducible at lower concentrations. Natively, E. coli inhabits the human gut where nutrients are often abundant, hence the membrane pores would preferentially be the ones with a smaller pore diameter, i.e. OmpC. Hence OmpF naturally may have a narrow range of functionality at extremely low osmolarity.

In both Figures 3 and 4, we observe a two fold difference in intensity between lowest and highest osmolarity. eGFP is seen to increase from nearly 10000 to 20000 AU while mRFP1 drops from 1600 to 800 AU. This trend is indicative of the activity of promoters and supports our hypothesis that OmpC promoter is activated under high osmolarity conditions, while OmpF promoter is activated at low osmolarity environment.

One important thing to note here is that the mRFP1 intensity is significantly lower than that of eGFP. Perhaps the fluorescent protein is not as strong in intensity as eGFP or perhaps the promoter OmpF is far weaker than OmpC. In order to characterise the functionality of individual promoters, we conducted further experiments to check the activity of promoters under different concentrations of solute.

Fluorescent Measurement of mRFP1 + PompC_mRFP1 Antisense Two constructs, one with constitutively expressed mRFP1 and one with constitutively expressed mRFP1 with PompC driven antisense were measured for their fluorescence using a microplate fluorescence reader. Each data point is an average of 4 readings — 2 biological and 2 technical replicates. All data is normalised to cell number by dividing arbitrary units of fluorescence by the respective OD600 absorbance value.

Figure 5: Fluorescence Intensity of constitutively expressed mRFP1 and mRFP1 with PompC regulated antisense RNA

We observe that the construct containing the antisense RNA against the constitutively expressed fluorophore has lower fluorescence intensity. Although fluorescence intensity of constitutively expressed mRFP1 is fairly stable in different Na+ concentration media, we cannot directly draw a relationship between the fluorescence of constructs with or without the antisense RNA. Therefore, we take the difference between the intensities to evaluate the promoter OmpC functionality.

Figure 6: PompC functionality with increasing osmolarity in the antisense RNA assay

From this chart it is evident that with increasing osmolarity, the difference in fluorescence increases, up till an intracellular osmolarity of close to 50mOsm/kg. This occurs at a medium concentration of 0.256M. Since PompC is active at higher osmolarities, we conclude that the fluorescence intensity difference is larger due to more antisense RNA being produced at high osmolarity of the medium. However, at an even higher osmolarity, the difference seems to shrink. This could be due to the cellular stress at such a high osmolarity. However, in TCS basal (Figure 3) it was noted that eGFP fluorescence which was regulated under PompC was still increasing. Regardless, we could still observe that the OmpC promoter functions to produce the antisense RNA in a osmolarity-dependent way.

There must be some limitation in the assay since in most other previously conducted studies where a reporter protein such as GFP (BBa_E0040) is regulated under OmpC promoter, there is an increasing trend with medium osmolarity [2], just as observed in TCS basal results (Figure 3).

Fluorescent Measure of eGFP + PompF_ eGFP Antisense Two constructs, one with constitutive eGFP and one with constitutive eGFP along with an antisense to eGFP controlled by the OmpF promoter were tested for fluorescence. We collected data from 2 biological replicates with 2 technical replicates. All data has been normalised with the OD to obtain intensity per fixed number of cells. We would expect the construct with antisense to have a lower fluorescence intensity. Moreover, since the antisense is controlled by the OmpF promoter, the intensity of fluorescence should show an increase with increasing medium osmolarity as OmpF is only activated at low concentrations.

Figure 7: Fluorescence Intensity of constitutively expressed eGFP and PompF regulated antisense RNA

The antisense RNA when inserted along with constitutively expressed eGFP significantly reduces the fluorescent intensity. The Hfq domain added to the antisense fragment may play a role in the enhancement of signal suppression but we need to set up controls without the domain in order to test it.

This also gives a qualitative measure of the activity of the promoter OmpF (PompF). We could infer that the promoter does function. The activity of this promoter is unlikely to be influenced by the strong promoter adjacent to it since the constructs are separated by a double terminator (Bba_B0015) and an AT rich 20bp segment. However, it appears that the fluorescence intensity of eGFP-antisense construct is comparable across the entire range of medium osmolarities. This indicates that PompF functions equally over different ranges of osmolarity. This contradicts our hypothesis since the antisense fragment would theoretically not be expressed at higher medium osmolarity.

If PompF is indeed that powerful a promoter, then the low fluorescent intensity of mRFP1 in the TCS basal construct is peculiar. Moreover, there is a decreasing trend in the TCS basal construct which supports the hypothesis. It could be possible that OmpC is influencing the activity of its reciprocal gene OmpF, either functionally or due to secondary structure of DNA. In order to test it, we measure the fluorescence from only PompF driven mRFP without PompC.

Improvements

The assay does present some limitations since the cellular differences between the two samples should also be taken into account. We could place the constitutively expressed eGFP and PompF controlled antisense RNA to eGFP in 2 different plasmids. We could then measure the fluorescence intensity from the same biological sample but in a condition with or without the antisense RNA.

Characterization of Negative Regulator Region of PompF The PompF that we used in our assays was taken from E. coli genome, -400 region from the OmpF ORF. This sequence contained the positive regulators F1, F2, and F3 as well as the negative regulator region labelled F4. This was confirmed from the paper by Yoshida et al. (2006). We wished to analyse what effect the F4 region has on the activity of the promoter. We hypothesised that with the negative regulator, the switch between OmpC and OmpF promoters would be more rapid and distinct.

We deleted the negative regulator region from the OmpF promoter via PCR and observed its activity by regulating a reporter mRFP1 under it. This was compared to the full OmpF promoter with the F4 region.

Figure 8: PompF (with negative regulator F4) working efficiency with increasing intracellular osmolarity

Figure 9: PompF (without negative regulator F4) working efficiency with increasing intracellular osmolarity

We observe that when the PompF promoter does not have the negative regulator, the decline in promoter activity is not as significant at an increasing osmolarity as when F4 was present. There is only a 1.6 fold reduction in the promoter activity with no negative regulator as compared to the near 2 fold decline in activity with the negative regulator present. In the presence of the negative regulator region, there is also a steeper decline and it occurs at a lower intracellular osmolarity. From this we can conclude that the F4 region on the promoter makes the promoter more sensitive to an increasing osmolarity while also allowing a discrete ‘switch’ like behaviour between OmpF and OmpC promoters.

We do observe a higher fluorescence intensity from when we remove the negative regulator region. Even though this is more desirable for our circuit, we still need the switch-like response from the promoter in order to get discrete red and green colours upon shellfish testing. We may need to seek to mutate some regions within the positive regulators of PompF to enhance its activity, make it work over a broader range of intracellular osmolarity and maintain its ‘switch-like behaviour’.

References:

1. "Part:Bba R0082 - Parts.Igem.Org". Parts.Igem.Org, 2019.

2. Yoshida, Takeshi et al. “Transcription regulation of ompF and ompC by a single transcription factor, OmpR.” The Journal of biological chemistry vol. 281,25 (2006): 17114-17123.

PROJECT

• Description
• Design
• Engineering
• Proof of Concept
• Implementation
• Safety
• Contribution
• Notebook

WETLAB

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• Results

DRYLAB

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REACH

• Human Practice
• Collaboration
• Communication
• Education
• Sustainable

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