Part:BBa_K2906004
AmilCP with C-Terminal secretion signal (HylA)
From the coral Acropora millepora, AmilCP is a non-fluorescent GFP-like chromoprotein that exhibits a blue-purple colour to the naked eye (Alieva et al., 2008; Beltran Ramirez, 2010; Liljeruhm et al., 2018). This composite part is made of AmilCP (BBa_K592009) , with a hydrophobic tag (BBa_K2906007) and a C-Terminal secretion signal (HylA) (BBa_K554002) to secrete the protein into the cytoplasm. It is expressed by a pTet promoter (BBa_R0040). Below we discuss our reasoning behind these choices and our quantitative and qualitative findings. The protein does express colour but unfortunately, it was unable to secrete.
Contents
HylA Signal Peptide
α-hemolysin has been previously characterised to provide efficient secretion of active correctly folded proteins to the culture supernatant in E. coli (Fraile et al., 2004). This hemolysin secretion system is a well-characterised type I secretion system which provides a one-step translocation, from the bacterial cytoplasm to the extracellular medium without a periplasmic intermediate of tagged proteins. The secretion signal does not become cleaved after trafficking across the bacterial membrane (Gentschev, Dietrich and Goebel, 2002; Ruano-Gallego et al., 2019).
Hydrophobic Tag:
Since we aimed at the creation of new hair dyes that would not damage the cuticle of hair, we did not want our designed coloured protein-based dyes to infiltrate the cuticle as this will lead to cuticle opening and weaken the hair itself. Therefore, both of our secreted variants also contained a novel hydrophobic tag (BBa_K2906007)
Characterisation:
To show whether this part worked, we decided to measure specific absorbance measurements at discrete wavelengths. This was done at 600 nm to measure optical density and bacterial growth, and at 588 nm because it has been previously shown to be peak absorbance of AmilCP (Alieva et al., 2008). For all figures, individual measurements were plotted. Additionally, we decided to perform a full spectrum analysis for each construct in order to measure the maximum absorbance. All the values plotted were previously processed by blank-correction. This procedure was done twice, the first time the cells were induced with 100 nM of anhydrotetracycline when their OD600 reached 0.4, and the second time when the OD600 reached 0.6.
Figures 1&2. Whole spectra reading. The full-spectrum analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. Measurements were performed at one-hour intervals for four hours, and then overnight. Curvature around 580-600 nm shows that there is some AmilCP production. The arrow in the first plot shows absorbance maxima, while the second plot did not show any clear peaks.
Figure 3&4. Whole spectra reading. The full-spectrum analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. Measurements were performed at one-hour intervals for four hours, and then overnight. Some curvature around 580-600 nm shows that there is some AmilCP production. Arrows on both plots show clear maxima for AmilCP absorption.
Figure 5&6. The above plots suggest that discrete measurements of E. coli cultures at 588 nm are not an accurate representation of AmilCP production in bacteria. As we can see in the spectrum scan, the same colonies could be easily shown to produce, or not to produce AmilCP. This might be because 588 nm is very close to 600 nm, the wavelength at which the optical density of E. coli is measured. Therefore there is interference and noise when measured at a discrete wavelength. The best way to measure AmilCP production is by spectral scan and seeing the relative increase in the absorbance.
Qualitative Data:
Colour: The image below shows the visible colour of the pellet obtained under normal light and UV light. This was done at five different time points after induction with 100 nM of anhydrotetracycline. AmilCP with HylA is labelled ''C-Terminal'' and can be seen present in both DH5a and BL21.
References:
Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M. E., Beltran-Ramirez, V., Miller, D. J., Wiedenmann, J., Salih, A., et al. (2008) ‘Diversity and evolution of coral fluorescent proteins’, PLoS ONE, 3(7). doi: 10.1371/journal.pone.0002680.
Beltran Ramirez, V. (2010) Molecular aspects of the fluorescent protein homologues in Acropora millepora. PhD thesis. James Cook University. Available at: http://eprints.jcu.edu.au/8837.
Fraile, S., Muñoz, A., De Lorenzo, V. and Fernández, L. A. (2004) ‘Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coli’, Molecular Microbiology, 53(4), pp. 1109–1121. doi: 10.1111/j.1365-2958.2004.04205.x.
Gentschev, I., Dietrich, G. and Goebel, W. (2002) ‘The E. coli alpha-hemolysin secretion system and its use in vaccine development.’, Trends in microbiology, 10(1), pp. 39–45. doi: 10.1016/s0966-842x(01)02259-4.
Ruano-Gallego, D., Fraile, S., Gutierrez, C. and Fernández, L. Á. (2019) ‘Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system’, Microbial Cell Factories. BioMed Central Ltd., 18(1). doi: 10.1186/s12934-019-1094-0.
Sequence and Features
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- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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