Part:BBa_K2906006
mRFP1 with C-Terminal secretion signal (HylA)
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 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. This part was able to express colour and was successfully secreted into the cytoplasm.
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 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 C-Terminal construct shows good levels of fluorescence expression in both DH5a and BL21 (DE3), showing that the protein is folding correctly.
SDS:
We also wanted to visualise if the protein was being overexpressed and secreted across the bacterial membrane. For that reason, a 5 mL sample was obtained both at time 0 before induction, and following induction and overnight incubation at 30ºC. The expected band size for mRFP1 with HylA is 31.6 kDa. After performing the SDS and running the gels, we noticed there was some background present, suggesting that there might be cells in the supernatant. To overcome this, and to prove secretion of our construct, we decided to filter-sterilise the supernatant by passing it through a 0.2-micron filter.
We can confidently conclude that the construct is expressing colour (see quantitative figures below) and is being successfully secreted into the media.
Qualitative Data:
Fluorescence Microscopy: After quantifying the data for mRFP1 we decided to perform fluorescence microscopy. After quantifying the data for sfGFP we decided to perform fluorescence microscopy. This would allow us to see the percentage of bacteria in a sample producing colour, 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 HylA (BBa_K2906006) showing some fluorescence, and BBa_K092300, which clearly expresses mRFP1.
Figure 3. 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 + HylA (BBa_K2906006) showing some fluorescence and low background noise, this shows that fluorescence is significant. 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 HylA is labelled ''C-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.
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.
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.
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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