Part:BBa_K2906005
mRFP1 with N-Terminal secretion signal (OmpA)
mRFP1 or monomeric RFP (also referred to EngRFP) is a red fluorescent protein which was first published in 2002 by Campebell et al. (2002). The coloured protein is derived from Discosoma sp. Despite mRFP1 being a monomer it was derived from the dimeric DsRed following multiple mutations. Although mRFP1 has a lower extinction coefficient, quantum yield, and photostability than DsRed, its rapid maturation (>10 times faster) shows similar brightness in living cells (Campbell et al., 2002). This composite part is made of mRFP1 BBa_E1010, 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 in 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 fluorescence, normalised by OD. Optical Density values were measured at 660 nm, this was done because it has been shown that OD660 gives a more accurate representation of bacterial growth in RFP-producing bacteria (Hecht et al., 2016). We grew the cells to an OD660 of ~0.6 and induced them with 100 nM of anhydrotetracycline. Then, they were loaded in triplicates into a 96-well plate and left overnight. For OD values, the blank was LB media, and for RFU the blank was E. coli TOP10 since it does not express any colour. The values were individually normalised by dividing RFU/OD and then averaged to plot the mean against time. An RFU value of 0 corresponds to baseline E. coli TOP10 measurements.
Figure 1. The plot shows the mean RFU/OD from three replicates of each construct expressed in E. coli DH5⍺ and BL21(DE3). The OD was measured at 660 nm and RFP fluorescence was measured at Ex ƛ 574, Em ƛ 618, every 15 minutes for 13 hours. The RFU values were normalised by the OD and the triplicates averaged. All values have been blank-corrected. A total of 52 recordings were made per well, with three wells per construct. The N-Terminal construct clearly shows no fluorescence values.
Qualitative Data:
Fluorescence Microscopy: After quantifying the data for mRFP1 we decided to perform fluorescence microscopy. We did this since the experiments above show that there is a trace amount of mRFP1 detected through fluorescence spectrometry, but no colour was seen. This would allow us to see the percentage of bacteria in a sample producing colour, if any, as well as some phenotypic characteristics of the bacteria with the construct. Below you can see three images, the negative control, which was E. coli TOP10, showing very limited background RFP fluorescence, mRFP1 with OmpA (BBa_K2906005) showing very limited amounts of fluorescence, and BBa_K092300, which clearly expresses mRFP1.
Figure 2. Fluorescent microscopy overlaid images of phase contrast and RFP filter. a) Negative control (E. coli TOP10) showing very slight background readings for fluorescence. b) mRFP1 + OmpA (BBa_K2906005) showing very limited fluorescence in two bacteria and high background noise, this shows that fluorescence is not intense. c) BBa_K092300 showing appropriate levels of fluorescence in most bacteria. The images above are composite overlaid images made from phase contrast and GFP-filter captures. They were processed using ImageJ.
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. mRFP1 with OmpA is labelled ''N-Terminal'' and can be seen present in both DH5a and BL21.
References:
Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A. and Tsien, R. Y. (2002) ‘A monomeric red fluorescent protein’, Proceedings of the National Academy of Sciences of the United States of America, 99(12), pp. 7877–7882. doi: 10.1073/pnas.082243699.
Hecht, A., Endy, D., Salit, M. and Munson, M. S. (2016) ‘When Wavelengths Collide: Bias in Cell Abundance Measurements Due to Expressed Fluorescent Proteins’, ACS Synthetic Biology. American Chemical Society, 5(9), pp. 1024–1027. doi: 10.1021/acssynbio.6b00072.
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.
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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