Part:BBa_K5057009

mCherry with the extracellular signal peptide HSTII

mCherry is a very rapidly-maturing monomeric red fluorescent protein with low acid sensitivity.[1]. The DNA sequence of this biobrick encodes a version of mCherry with an additional sequence designed for liquid-liquid phase separation purposes and an extracellular localization sequence.

Usage and Biology

For the characterization of different composite parts (Biobrick:) we needed to verify the functionality of the signaling sequence HSTII. We decided to fuse HSTII with mCherry enabling an easy verification method via fluorescence measurement. Since various proteins could have different effects on bacterial growth and require different conditions to be efficiently expressed,additionally, we aimed to determine the optimal IPTG concentration and temperature for induction.

Initial tests with E. coli BL21(DE3) showed high basal expression of HSTII-mCherry without induction (data not shown), making it difficult to properly characterize. Therefore, we chose to continue with E. coli BL21(DE3) containing a pRARE2 LysS plasmid. This plasmid encodes a lysozyme which binds to the T7 polymerase leading to inhibited basal expression, helping us with the characterization of HSTII-mCherry. E. coli BL21(DE3) pRARE2 LysS cells were transformed with pET-HSTII-mCherry. The cultures were incubated in 700 mL MH- medium containing Ampicillin and Chloramphenicol (MH-Amp-Cm) at 37°C to an OD600 of 0.6. Four different concentrations of IPTG (0.1 mM, 0.5 mM, 0.7 mM, 1.0 mM) were tested at three different temperatures (18°C, 25°C, 37°C) in 20 mL of culture. As a control, an uninduced culture was used. Expression of mCherry was determined by fluorescence measurements of pellets and supernatants. After 4, 12 or 24 hours, depending on the temperature, 3x 1 mL per culture was pelleted and diluted to an OD600 of 1.0. The pellets were washed two times with phosphate-buffered saline (PBS). The supernatants (SN) were directly used for fluorescent measurement. Fluorescent measurements were carried out as technical duplicates using a plate reader at an excitation of 570 nm.

Fig. 1: Experimental Set-UpTest of different induction conditions for pET-HSTII-mCherry in E. coli BL21(DE3) with pRARE2 LysS plasmid. Uninduced cultures were used as a negative control.

Induced samples consistently showed higher fluorescence compared to uninduced samples, indicating successful induction in most conditions. The low fluorescence in uninduced cultures indicates minimal basal expression from the T7 promoter. However, since the difference between uninduced and induced cultures is notable, therefore IPTG can be used for inducible expression reliably. All supernatants showed a fluorescence intensity which is approximately five-fold lower compared to pellets, which is due to the fact that the proteins are less concentrated in the supernatant compared to the pellet. Temperature had a significant impact on induction efficacy. The lowest signal was measured in cultures incubated at 37°C, suggesting minimal or no induction of mCherry expression at this temperature. For cultures at 30°C, only minimal differences between applied IPTG concentrations were observed. The fluorescent signal in the pellet was comparable to the uninduced sample, while the signal of the induced supernatant was slightly increased. The overall highest fluorescence in both pellet and supernatant compared to uninduced control was observed for samples incubated at 18°C and measured after 24 hours. Induction with 0.1 mM IPTG yielded higher fluorescent signals compared to other concentrations. Therefore, for the verification of the functionality of HSTII we used the same conditions.

Fig. 2: Induction conditions of E. coli BL21(DE3) pRARE2 LysS cells containing pET-HSTII-mCherry at different temperatures and IPTG concentrations. HSTII-mCherry gene expression was induced with different concentrations of IPTG (0 mM, 0.1 mM, 0.5 mM, 0.7 mM, 1.0 mM) at different temperatures (18°C, 30°C, 37°C) in MH-medium. Fluorescence of A: Pellet and B: Supernatant (SN) was measured with a plate reader at an excitation of 570 nm. The experiment was performed in biological duplicates, each consisting of technical duplicates that were individually measured three times. Pellets were washed with Phosphate-buffered saline (PBS) and the OD600 was normalized for all cultures before measurement.

Although in the previous experiment, differences in the fluorescence intensity of the differently induced supernatants were noticeable, we could not exclude that the measured signal could be caused by proteins released in the medium through dying bacteria or handling of the samples. Therefore, in the following experiment, we decided to compare the fluorescence between mCherry expressed with and without the signaling sequence. Therefore, E. coli BL21(DE3) with pRARE2 LysS plasmid were transformed with the pET-HSTII-mCherry and the pET-mCherry, respectively. The cultures were diluted to an OD600 of 0.1 and incubated in 30 mL MH-medium with Ampicillin and Chloramphenicol at 37°C to an OD600 of 0.6. The cultures were induced with 0.1 mM IPTG. After 24 hours at 18°C and 200 rpm continuous shaking, 2x 1 mL of the culture was pelleted and diluted to an OD600 of 1.0. The supernatants (SN) were stored for fluorescent measurement. The pellets were washed two times with phosphate-buffered saline (PBS). The pellets and the supernatants were used for fluorescent measurement using a plate reader with an excitation of 570 nm and emmision at 610 nm.

Fig. 2: Experimental Setup Expression of pET-HSTII-mCherry and pET-mCherry in E. coli BL21(DE3) with pRARE2 LysS plasmid.

The pellets of cultures expressing mCherry or HSTII-mCherry showed comparable levels of fluorescence (Fig. x). Conversely, supernatants from HSTII-mCherry culture exhibited much higher fluorescence compared to the supernatant of mCherry culture. The overall fluorescence in supernatants was lower than in pellets for both constructs, consistent with expected lower protein concentration in the extracellular medium. This comparative study between HSTII-mCherry and mCherry provided strong evidence for the functionality of the HSTII signal peptide. The higher fluorescence signal in the supernatant of HSTII-mCherry cultures compared to mCherry cultures suggests successful export of the fusion protein mediated by the HSTII signal peptide. The comparable fluorescence levels in the pellets indicate that the presence of the HSTII signal does not significantly affect overall mCherry expression or folding. These results validate our approach of using HSTII as a signal peptide for extracellular localization of our target proteins.

Fig. 4: Expression of mCherry and HSTII-mCherry in E.coli BL21(DE3) with pRARE2 LysS plasmid Cultures were induced with 0.1 mM IPTG at 18°C in MH-medium with Ampicillin and Chloramphenicol. Fluorescence of Pellet and Supernatant (SN) was measured using a plate reader at an excitation of 570 nm. Pellets were washed with Phosphate-buffered saline (PBS) and OD600 was normalized for all cultures before measurement. The experiment was performed in technical duplicates that were individually measured three times.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 780
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 798
  

References

[1] Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology. 2004 Nov 21;22(12):1567–72. ‌