Part:BBa_K2906003
AmilCP with N-Terminal secretion signal (OmpA)
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 an N-Terminal secretion signal (OmpA) BBa_K208003 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. Unfortunately, the protein was unable to show colour and to secrete.
Contents
OmpA signal peptide:
Outer membrane peptide A (OmpA) is a conserved protein domain found at the outer membrane of many gram-negative bacteria such as E. coli (Wang, 2002; Smith et al., 2007). The OmpA secretion signal is located at the N-terminal region of the protein of interest and becomes trafficked through a type II-dependent secretion system (Kotzsch et al., 2011). The signal peptide is cleaved by the secretion machinery in the plasma membrane of the bacterium. The resulting protein is secreted and released in an active mature form. This secretion signal peptide was therefore selected due to its reposted high efficiency of secretion during high protein expression (Pechsrichuang et al., 2016).
Hydrophobic Tag:
Since we aimed at the creation of new hair dyes that would not damage the cortex of hair, we did not want our designed coloured protein-based dyes to infiltrate the cortex 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. No curvature around 580-600 nm shows that there is no AmilCP production. Bacterial cultures seem to be growing slower suggesting possible toxic protein aggregates.
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. No curvature around 580-600 nm shows that there is no AmilCP production. Bacterial cultures seem to be growing slower suggesting possible toxic metabolites.
Figure 5&6. The N-terminal transformed bacteria seem to decline over time in the induced samples at OD600 ~0.4. This is true in E. coli DH5⍺ and BL21(DE3). While this is not equally as visible in replicate 2 (when cells were induced at OD600 of ~0.6) we can, however, highlight a degree of stunted growth. From the growth determination, we can conclude that the AmilCP N-terminal part must be causing toxic aggregates in the E. coli cells.
Figure 7&8. 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.
Protein Expression
Based on our SDS-PAGE results we can conclude that AmilCP over-expression was not fully obtained. The AmilCP N-terminal expression is only obtained in replicate 1 when cells were induced at an OD600 of 0.4 (figure above). This suggests that the protein is being produced but it is aggregating in the inclusion bodies. No secretion was detected.
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 OmpA is labelled ''N-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.
Kotzsch, A., Vernet, E., Hammarström, M., Berthelsen, J., Weigelt, J., Gräslund, S. and Sundström, M. (2011) ‘A secretory system for bacterial production of high-profile protein targets’, Protein Science, 20(3), pp. 597–609. doi: 10.1002/pro.593.
Liljeruhm, J., Funk, S. K., Tietscher, S., Edlund, A. D., Jamal, S., Wistrand-Yuen, P., Dyrhage, K., Gynnå, A., Ivermark, K., et al. (2018) ‘Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology’, Journal of Biological Engineering. BioMed Central Ltd., 12(1). doi: 10.1186/s13036-018-0100-0.
Pechsrichuang, P., Songsiriritthigul, C., Haltrich, D., Roytrakul, S., Namvijtr, P., Bonaparte, N. and Yamabhai, M. (2016) ‘OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system’, SpringerPlus. SpringerOpen, 5(1). doi: 10.1186/s40064-016-2893-y.
Smith, S. G. J., Mahon, V., Lambert, M. A. and Fagan, R. P. (2007) ‘A molecular Swiss army knife: OmpA structure, function and expression.’, FEMS microbiology letters, 273(1), pp. 1–11. doi: 10.1111/j.1574-6968.2007.00778.x.
Wang, Y. (2002) ‘The function of OmpA in Escherichia coli’, Biochemical and Biophysical Research Communications, 292(2), pp. 396–401. doi: 10.1006/bbrc.2002.6657.
Sequence and Features
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