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Characterisation of LasI by Shanghaitech iGEM 2017

Group: Shanghaitech 2017

1、LasI coding sequence is functional

Firstly, to test whether BBa_C0178 - LasI coding sequence is still functional, we directly detected Las molecule by HPLC and LC-Mass Spectra, which is product of LasI’s enzymatic reaction. The results are shown in Fig.1.

Figure 1: LasI is functional

2、LasI coding sequence can be used to design complex logical circuits (like signal transfer by converter)

As for our achievement, ‘Rpa-Las molecule converter’ can achieve a function – signal transfer. Fig. 2 shows our idea. We can build up different blocks including different converter devices (Rhl-Lux, Lux-Rpa, Rpa-Las, Las-Tra, Tra-Sin and so on). These blocks can form a more complex signal transfer system. This year we have successfully constructed ‘Rpa-Las molecule converter’ BBa_K2315046, indicating in principle our multiple layer signal transfer would work.

Figure 2: Signal transfer system using converters

3、Las molecule 3OC12 can be quantitatively detected by HPLC and LC-MS

It is generally hard to directly measure small chemical signals such as AHL.s due to their low molecular weights compared to proteins. We decided to use advanced methods to measure Las. We succeeded in using High Performance Liquid Chromatography (HPLC) and Liquid chromatography–mass spectrometry (LC-MS) to detect Las 3OC12-HSL. Figure 3 shows our results of two devices - Rpa-Las molecule converter BBa_K2315046 and Las molecule generator BBa_K2315033. Also, the spike areas can be used to quantitatively estimate the concentration of Las as shown in the upper right corner of the figures. We found the relative peak area is proportional to the concentration (Fig.3-2a). The correlation coefficient is very high (0.9964, Fig.3-2b).

Figure 3-1: Rpa-Las converter

Figure 3-2: Las molecule generator

4、Las has a long half-life, very stable in culture mixture

If Las molecule can be generated, is it stable? What’s its half-life time? To test the half-life, we did the following experiment. We collected the supernatant from Las generator bacteria with BBa_K2315033, and then measure its time lapse concentration using HPLC every hour for 7 hours (Fig.4a). We also verified the molecular integrity by LC-MS (Fig. 4b). With time lapse, the relative pike area decreases slowly. Yet, all values have the same magnitude (10E8), which suggests that Las molecule is stable. The half-life is estimated to be > 6h. So Las molecule has a high stability and very robust.

Figure 4: Las molecule attenuation

5、QS systems’ AHL concentration (here is Las molecule – 3OC12 HSL) can be calibrated by HPLC’s relative peak area.

We use the Las molecule bought from Adipogen to make samples for testing.

Figure 5: Las molecule standard curve

• a) HPLC results for 4 different samples with a Las molecule concentration gradient. The relative peak area shows the amount of Las molecule.
• b) Standard curve made by origin.
• c) LC-MS result of LasI product

According to the standard curve above, the R^2 value is close to 1, which means that the curve fitting is successful. In conclusion, we can analyze the Las molecule amount by HPLC with standard curve.

6、Las concentration can be estimated by GFP fluorescence by plate reader and fluorescence microscope

For demonstrating GFP expression of Las molecule reporter BBa_K2315034, we used fluorescence microscope to observe the GFP’s green fluorescence. Figure 6 shows two different samples – one was added the Las molecule generator's supernatant and another wasn’t. Between Negative control and Positive control under dark field, we can clearly distinguish the fluorescence’s difference – Positive control is much brighter than Negative control. Thus, LasI’s product can be calibrated by GFP’s fluorescence by plate reader and fluorescence microscope by Las reporter.

Figure 6: Las reporter GFP expression under fluorescence microscope. Positive control is with Las generator (LasI+) bacteria supernatant . Negative control does not contain the LasI+ supernatant.

Furthermore, we also test devices by plate reader. More information can be seen in these parts: BBa_K2315046 BBa_K2315034.

Applications of BBa_C0178

Characterization of BBa_C0178-Arizona_State 2016

Authors: Ernesto Luna, Brady Dennison, Cassandra Barrett, Jimmy Xu, Jiaqi Wu, Dr. Karmella Haynes

Our team helped increase characterization of the part Bba_C0178(LasI). This part was tested against its ability to induce the part BBa_F2620 by the Canton Lab(MIT). This part outputs PoPS as a Receiver Device combined with LuxR. An induction test on BBa_F2620 had been done by Dr. Barry Canton (2008), but they tested GFP production over various AHL concentrations, while our test was an 8-hour GFP read over time for 2 AHL concentrations (10 and 50%). In addition, the Canton test utilized synthetic AHLs while our test utilized AHLs produced via an E.coli chassis. A visual induction test was also done, plating the Sender alongside a GFP positive control, negative receiver control, and F2620.

As shown below, Las was unable to induce F2620 in this visual induction, as colonies in the top right section did not produce GFP. This is not the expected result, since the Canton Lab showed that the Las AHL (3-oxo-C12-HSL) was capable of inducing F2620. This may have been due to an issue with AHL diffusion on the plate and will be examined more in the plate reader test.

The figure below compares LasI at 10% and 50% concentrations alongside the native AHL system LuxI at 10% and 50% concentrations. LasI is shown to induce F2620, but to a lesser degree than LuxI. This affirms that F2620 is capable of being induced by LasI synthesized within BL21(DE3) E. coli, supporting the notion that crosstalk is occurring. This result contrasts with the plate induction result, but because it is supported by the Canton results, it is likely that the plate induction for LasI was erroneous.

AHL Disposal Test

The final experiment conducted using this part aimed to determine proper safe disposal procedures for the 3-O-C12-HSL. This AHL molecule is capable of crosstalk with potentially pathogenic strains of bacteria, and proper disposal of these AHLs should be an important biosafety measure taken. S.A. Borchardt had already tested the susceptibility of AHLs to bleach and found that 3-oxo AHLs were easily broken down by bleach while other AHLs were not. Our experiment aimed to test the application of standard EH&S sanitation protocols on AHLs (10% bleach solution and autoclaving). The figure below indicates that AHLs produced by LasI were properly deactivated by a 10% bleach solution. This was the expected result, as LasI produces a 3-oxo AHL, which should have been destroyed by bleach.

A standard 15 minute Liquid autoclave cycle was also used to treat an extracted AHL solution. The figure below indicates that LasI was nearly completely destroyed via autoclaving. This was the expected result, as the high pressure and temperatures should deactivate any AHL molecules present.

Conclusion

The results demonstrate that Las was able to effectively induce F2620 after being extracted. The Las results were consistent, which showed significantly decreased induction when treated with bleach, indicating complete AHL inactivation. According to the autoclave results, a standard 15 min liquid procedure is able to degrade nearly all AHLs. The extreme pressure and temperature generated by the autoclave was more than enough to remove any threat posed by these AHL samples. In summary, our data suggests that, for LasI, both bleach and autoclaving are capable of deactivating 3-oxo-C12-HSL.

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