Team:Uppsala Universitet/Contribution

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tsPurple purification results

We expressed tsPurple (BBa_K1033906) with a his-tag in E. coli DH5α cells using the high copy pSB1C3 vector with a BBa_J23110 promoter. We selected and cultured two different tsPurple expressing colonies, lysed them, and used affinity chromatography with Ni-NTA-agarose to purify two separate samples of his-tsPurple.

After purification, the protein was seen as a band at a size of approximately 28 kDa on 10% SDS-PAGE gels of both samples (Figure 1.). Considering that the his-tag adds roughly 2.5 kDa [1] to the total weight of the protein, we can conclude that tsPurple’s molecular weight is around 25.5 kDa, this result is consistent with SVCE_Chennai iGEM-2016 team’s computational data.

**Figure 1: SDS-PAGE analysis of purification of tsPurple sample 2.**SDS-PAGE analysis of affinity purification steps of the His-tagged protein from *E. coli* cell lysate, flow-through (F), wash 1 (W1), wash 2 (W2), elutions 1-4 (E1-E4), and elute 3 after dialysis for buffer exchange (D) on 10% SDS-PAGE stained with Coomassie Blue. Purified tsPurple is seen as a band at a size of approximately 28 kDa.

We used Bradford assay to determine protein concentration in both samples and measured their fluorescence spectra. For absorption spectrum, we concentrated the protein samples 10x with Amicon Ultra-0.5 mL Centrifugal Filters (3K) to get better absorption peaks.

tsPurple absorbance, excitation and emission spectra

Excitation is equivalent to absorption as it brings a molecule to its excited state S_n. Thus when there is no interference from impurities in the solution, the excitation and the absorption spectra of a fluorophore should be identical [2]. Our results showed, indeed, that both spectra have their peaks at 588 nm, while the emission peak was at 624 nm (when excited at 540 nm) (Figure 2a, 2b, 2c).

We chose a LacI fused cy3 fluorophore as our positive control for fluorescence measurement. Cy3 has an excitation peak at 550 nm and an emission peak at 570 nm [3], the closest to tsPurple’s peaks among all the fluorophores that we had on hand. Through absorbance measurements at 540 nm, we normalized LacI-cy3 and tsPurple’s concentrations to the same value: Abs₅₄₀ = 0.03. As can be seen from the comparison in Figure 2d, tsPurple is a non-fluorescent protein.

**Figure 2: Excitation (a), absorbance (b) and emission (c) spectra of tsPurple; Emission spectra of tsPurple vs. LacI-cy3 (d).**Fluorescence measurements were done using the microplate reader model Tecan Spark 10M in a flat black 384, low volume plate with non-binding surface. The measurements were made in a range of 440-750 nm. Absorbance measurements were done using Nanodrop 2000 (UV-vis), blanked against PBS buffer, in a range of 200-750 nm.

Manganese perosidase expression in Pichia pastoris

To express BBa_K500001, overhang PCR was performed to add complementary regions towards the commercial pPICZαB vector, in which MnP was cloned by gibson assembly. In this vector, the gene is controlled by the AOX1-promoter and is cloned in-frame to an N-terminal α-secretion factor from S. cerevisiae. MnP was expressed in the X-33 wildtype strain of Pichia pastoris, where secretion was observed (Figure 3).

The full expression circuit can be found here: BBa_K3105682

**Figure 3: Expression and secretion of Manganese Peroxidase (MnP).** X-33 P. pastoris cells were transformed with pPICZαB_MnP, induced (i), fractionated into pellets (P) and supernatants (S), and analysed by 10 % SDS-PAGE stained with Coomassie Blue. u, uninduced Induction bands are visible at approximately 70 kDa for both P and S (red arrows), showing secretion of MnP.

His-tsPurple was first made and published by Josefine L. et al. from Anthony Foster's group at Uppsala University, Sweden [4]. We would like to thank Josefine and Anthony for kindly providing us with the his-tsPurple plasmid.

References

[1] Booth, William T., et al. “Impact of an N-Terminal Polyhistidine Tag on Protein Thermal Stability.” ACS Omega, vol. 3, no. 1, Jan. 2018, pp. 760–68, doi:10.1021/acsomega.7b01598.

[2] Albani, J. R. “Chapter 2 - Fluorescence: Principles and Observables.” Structure and Dynamics of Macromolecules: Absorption and Fluorescence Studies, edited by J.R. Albani, Elsevier Science, 2004, pp. 55–98, doi:10.1016/B978-044451449-3/50002-2.

[3] Abcam®, “A Guide to Fluorochromes”. Online, accessed on 2019-08-19. https://docs.abcam.com/pdf/immunology/fluorochrome_guide.pdf.

[4] Liljeruhm, Josefine et al. “Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.” Journal of biological engineering vol. 12 8. 10 May. 2018, doi:10.1186/s13036-018-0100-0.