Protocols

Transformation of plasmid into Salmonella Typhimurium bacteria

The transformation procedure is executed through a heat shock method. This approach is adapted from Central Laboratory of Virology and Cancer Pathobiology Research Medicine Faculty Universitas Indonesia

This procedure is applied during Eira Cyan Fluorescent Protein (CFP) transformation at Salmonella Typhimurium ATCC 14028, and buforin IIb along with p53 sensor incorporation to A1-R

• First of all, Salmonella Typhimurium culture that has been grown at liquid medium culture for minimal 12 hours will be incubated at ice for 1 hour.
• After that, the liquid culture will be centrifuged at 4000 rpm for 10 minutes. The obtained pellet will be incubated further under a 20 μL kalium rich transformation buffer in ice for 1 hour.
• For the next step, the culture will be centrifuged again at 4000 rpm for 10 minutes. The generated pellet will be incubated at a 20 μL calcium-rich transformation buffer in ice for 15 minutes.
• The liquid suspension will be centrifuged again at 4000 rpm for 10 minutes. The obtained pellet will be mixed with a 200 μL calcium-rich transformation buffer, then mixed thoroughly with pipetting. This fluid suspension will be stored on ice.
• Altogether with other components (plasmid diluted with 60 μL nuclease-free water (NFW) incubated for 10 minutes), the required materials (consisting of 5 μL plasmid, 50 μL processed Salmonella suspension culture, and Eppendorf tube) will also be incubated on ice for 30 minutes.
• These materials will be soaked in hot bathwater at a temperature of 38℃ for 90 seconds, then incubated on ice for 1 minute.
• The later liquid suspension will be added to 200 μL Super Optimal Broth with Catabolite Repression (SOC), then incubated for 1 hour at 37℃ at a speed of 150 rpm.
• 100 μL final culture will then be spread evenly through ATCC 3 agar. The final product can be obtained after 16 hours of incubation.

Mutation Salmonella Typhimurium into A1-R strain

The mutation is adapted from previous study, as stated by Zhao, et al. [12]

• First of all, EiraCFP will be transformed into Salmonella Typhimurium bacteria. Please refer to the procedure above regarding further details of transformation.
• 27 μL of transformed Salmonella Typhimurium liquid culture will be centrifuged at 4℃ with 3500 rpm. The obtained pellet will be mixed with N-methyl-N-Nitro-N-Nitrosoguanidine (NTG) to obtain random mutation. The prepared NTG is obtained through diluting 3 mg NTG with 3 ml sterile aquadest.
• The mixture will be added with a 24 ml tris-maleate buffer that has been manufactured at pH 6. The liquid suspension will then be vortexed prior to 30 minutes incubation.
• The bacteria liquid culture will be streaked on ATCC 3 agar and left for a minimum of 12 hours.
• After that, the grown bacteria colony will be selected through Ames test. A1 colony will be attracted to leucine and arginine compounds, and able to grow in high numbers (with a minimum of 12 hours incubation).
• For the next step, Salmonella Typhimurium A1 strain will be selected further with the HT-29 cell line. 10 ml bacteria strain will be added to 104 HT-29 cell line. This step will be repeated for 3 days, where bacteria strain will be continuously subcultured along that duration.
• After that, the culture will be homogenized and added to phosphate-buffered saline (PBS) 1x.
• The homogenized liquid will go through centrifugation, and later the supernatant will be added in LB medium broth 1 hour prior to streak in LB agar. The culture will be incubated for 12 hours at 37℃.
• The grown culture will be checked under UV light with a wavelength of 458 nm. The glowing colony, indicating A1-R colony, will be retrieved and cultured further in LB medium.

Cell count (HeLa cell & CERVEX strain)

We performed cell counting with a hemocytometer. The dilution was made using 10 µl of Hela cell suspension and diluted with trypan blue in 1:2 dilution. The observation was conducted using electron microscope, and the death cell expressing trypan blue was counted using this formula:

In the assessment of Cerve-X bioavailability, we used Eira CFP with 458 nm excitation. The illuminated cells expressing GFP were captured using a camera, and imaging software is employed for quantifying fluorescence parameters.

Quantitative buforin secretion assessment

The secretion will be assessed through bicinchoninic acid after His-Tag purification, and will be further analyzed through SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)