Improvement of an Existing Part

Introduction

Bioluminescence is found in various branches of different species including fungi, metazonas and bacteria [1]. Amongst these, the most extensively studied and used is the luciferase from the North American firefly Photinus pyralis. The firefly luciferase is a bifunctional enzyme that catalyzes the oxidation of firefly D-luciferin to excited oxyluciferin in the presence of oxygen and ATP. This reaction leads to emittance of a yellow-green light in the range of 560 nm when the electrons of oxyluciferin return to their ground state. These properties make the Firefly luciferase useful as a reporter and is, among other things, used as a reporter for gene expression and promoter activity.

The luciferase of P. pyralis is a thermolabile enzyme with an in vitro half life of 2-3 minutes at 37°C [2]. This is an advantage in some cases of time-sensitive studies such as for induction or repression of gene expression and when testing response to toxins and infection. However, there would be benefits with a luciferase with higher thermostability and longer half-life in cases of studies that are not time-sensitive, such as colonization of metastases or monitoring of tumor growth. The increased thermal stability would lead to increased accumulation of luciferase and thus a higher light output and improved detection.

The study Firefly Luciferase Mutant with Enhanced Activity and Thermostability suggests the introduction of eight amino acid mutations in the wild-type luciferase to increase thermostability and catalytic activity [1]. These includes the mutations of T214A (ACT>GCC), A215L (GCC>CTG), I232A (ATT>GCC), F295L (TTC>CTG), E345K (GAG>AAA), I423L (ATA>CTG), D436G (GAC>GGC) and L530R (CTC>CGC). These are the mutations that are introduced to the original gene used in our experiments, where we compare the performance and thermostability of the original gene and the mutated version of the luciferase.

The parts that were tested, both the already existing and the mutated version, can be found under the iGEM parts-page. The original gene used in our experiments is the part originally designed by team iGEM07_Ljubljana, BBa_I712019 . The mutated version designed by us is the part BBa_K4367016 .

Results

The result of the first PCR of the integration vector is shown in figure 1 below. The expected length of the fragment is around 2200bps, which is consistent with the result of the gel electrophoresis. Unfortunately, the gel results for the PCR reactions of BBa_I712019 and BBa_K4367016 were not saved, but showed clear lines slightly above the fifth line of the DNA ladder, indicating a length of around 1600bps as expected.

Figure 1. The result from gel electrophoresis of the pYTK095 PCR product, indicating a length of above 2000bps.

The luminescence was examined using a plate reader at 25℃, 35℃ and 45℃. The results are shown in the bar chart in figure 2 below. The bar chart was constructed by taking the mean of the experimental data for each part. The raw data from the experiment and the process of constructing the bar chart is presented in Appendix A.

Figure 2. Bar chart showing the mean of the experimental data of each part and the negative control, for the three different temperatures.

In the first experiment with the temperature set to 25℃, the highest luminescence value was recorded for one of the samples of the original part BBa_I712019, at 2E6RLU. Luminescence was also recorded for all mutated parts BBa_K4367016, varying between approximately 150000-200000RLU and for a second colony of the original part at approximately 100000RLU. For the experiment at 35℃ the luminescence of the mutated part colonies is slightly higher compared to the 25℃ experiment, at around 260000-400000RLU. Luminescence drops to 70000RLU and 2000RLU for the two colonies of the original part showing luminescence. The last experiment at 45℃ shows a decreasing luminescence for all samples. However, luminescence for all four mutated part colonies is still showing, and the luminescence for the first colony of the original part (005) is quickly decreasing at this temperature.There is no luminescence recorded for any of the negative control samples.

Conclusion

The highest value of luminescence that is shown in the experiment of 25℃ is perhaps not to be considered as a reasonable value, the gain settings for the plate reader program could probably be altered to a slightly lower value to give a more reasonable output. It does, however, show luminescence and the fourth colony of the original part expresses luminescence at a lower level, more similar to the colonies of the mutated part, which is expected at this temperature. When the temperature is increased, the luminescence signal is decreasing for all samples but with a lower rate for the mutated part than for the original part. The fourth colony of the original part does not show any luminescence at 35℃ or at 45℃.

These results and the trend of the bar chart indicates that the mutated version of the Firefly luciferase is actually more thermostable and shows a higher catalytic activity than the original part. At a temperature of 45℃ the luminescence of both the mutated and original part drops quite drastically, but the luminescence is still showing clear luminescence and is considerably higher for the mutated part compared to the original. However, even if the results look promising, two colonies of the original part do not express luminescence, even at a temperature of 25°C. Due to time constraints we were not able to sequence the parts, which would ensure the correct assembly and integration into the cells. More experiments should therefore be carried out to better confirm the improvement of thermostability and catalytic activity of the improved part.

Protocol

The plasmid pYTK095, the original gene BBa_I712019 and the mutated gene BBa_K4367016 were each amplified with 50µl Phusion PCR according to the protocol in Appendix B. The PCR reactions were verified with gel electrophoresis. 1µl DpnI restriction enzyme was then added to the remaining solution of the PCR reaction and was then incubated at 37°C for 2hrs. The gene fragments were each assembled into the plasmid pYTK095, using Gibson Assembly according to protocol in Appendix C and transformed into E.coli according to the protocol in Appendix D. Four colonies from the plate with BBa_K4367016 and BBa_I712019 respectively were inoculated for overnight culture. After 14hrs, the OD was checked and all cultures were diluted to OD=1 with a final volume of 5ml. The diluted cells were spun down at 5000rcf for 5 minutes, the supernatant was removed and the pellet was resuspended with 5ml PBS. The resuspension was spun down again, supernatant was removed and pellet resuspended in 1ml PBS.

The lysis and luciferase assay was carried out according to the protocol in Appendix E. The parts and information about the parts, as well as primers used for PCR reactions can be found at the respective part pages for BBa_I712019 and BBa_K4367016 .

Appendix

Appendix A
Bar chart design and raw data

Appendix B
Phusion PCR protocol

Appendix C
Gibson Assembly protocol

Appendix D
E. coli transformation protocol

Appendix E
Luciferase Assay System protocol

References

[1] Pozzo T, Akter F, Nomura Y, Louie AY, Yokobayashi Y. Firefly luciferase mutant with enhanced activity and thermostability. ACS Omega [Internet]. 2018 [cited 2022Oct11];3(3):2628–33. Available from: https://doi.org/10.1021/acsomega.7b02068

[2] Baggett B, Roy R, Momen S, et al. Thermostability of Firefly Luciferases Affects Efficiency of Detection by in Vivo Bioluminescence. Molecular Imaging [Internet]. 2004 [cited 2022Oct11];3(4). Available from: https://doi.org/10.1162/15353500200403178