Background

Efficient and low cost production of valuable natural compounds, like proteins, has developed into a leading industry. Starting, by choosing a suitable production host followed by establishing a profitable downstream process, every step is constantly optimized to increase overall yields.

When it comes to choosing a production host, Bacillus subtilis is particularly interesting: the Gram-positive model organism can be easily genetically modified and has powerful secretion capacities. [1]

In this part of EncaBcillus we aimed at making use of the B. subtilis native advantages and combined them with Peptdiosomes – a new innovative cultivation platform for functional co-cultivation. Multiple Peptidosomes, with each encapsulating one specific strain that secrets one protein of interest. By doing so, the production of multi-complex proteins cloud be achieved by separated subpopulations all in one reaction hub.

To ensure the assembly of proteins outside of the Peptidosomes we further characterized the SpyTag/SpyCatcher system. Theses functional units can be attached to any protein of interest and upon secretion will result in a covalent isopeptide bond between the SpyTag/SpyCatcher partners [2]. The system originates from Streptococcus pyogenes and since it’s discovery it has been under constant development [3]. In our project we applied codon-adapted B. subtilis specific tags and reduced the SpyCatcher in length to enhance it´s usability when translationally fused to a protein of interest. Thus, decreasing the chances of the tag interfering with overall protein folding. [4]

To demonstrate the applicability of both tags we fused them to a green (sfGFP) and a red (mCherry) fluorescent protein, enabling an easy detectable output. (For more details please check our Design section) Since a core part of this project involves secretion, we included a signal peptide in front of all our constructs. (click here to learn more about our Signal Peptide Toolbox).

To evaluate the efficiency of the secretion process we monitored the fluorescence in supernatants harvested from B. subtilis strains carrying our constructs and compared them to supernatants obtained from the wild type. In order to prove the functionality of the SpyTag/SpyCatcher system we co-incubated supernatants derived from different strains and performed a SDS-Page where we could demonstrate the formation of a fusion protein.

Design

All of the composite parts necessary for the genetic constructs were equipped with the RFC 25 standard, cloned into pSB1C3 backbone and submitted to the registry. All cloning was done according to standard protocols and the plasmids were stored in Escherichia coli DH10β. All constructs were verified by sequencing.

The gene encoding the mini. SpyCatcher (BBa_K2273015) was chemically synthesized. The codon optimized SpyTag (BBa_K2273014) was generated via overlapping primers iG17P049 and G17P050 and amplified using the primers TM4487 and iG17P039. We used a sfGFP (BBa_K2273033) that was codon optimized for Streptococcus pneumoniae, which has been demonstrated to work best in Bacillus subtilis [5]. The used mCherry (BBa_K2273034) was codon adapted for B. subtilis (Popp et al., 2017, accepted). The His-tag, necessary for protein purification was included in the reverse primers (Table 1).

Gene	BioBrick	forward Primer	reverse Primer
SpyTag_His-tag	BBa_K2273016	TM4487	iG17P068
Mini. SpyCatcher_His-Tag	BBa_K2273017	TM4487	iG17P067
sfGFP_His-tag	BBa_K2273021	TM4487	iG17P065
mCherry_His-tag	BBa_K2273022	TM4487	iG17P066

In order to identify the best combination of SpyTag/SpyCatcher and FP fusion, we constructed all N- and C-terminal combinations (Table 2).

Table 2: List of SpyTag/SpyCatcher and FP composite parts. pSB1C3-mCherry-SpyTag-His BBa_K2273035 pSB1C3-mCherry-SpyCatcher-His BBa_K2273036 pSB1C3-SpyTag-mCherry-His BBa_K2273037 pSB1C3-SpyCatcher-mCherry-His BBa_K2273038 psB1C3-sfGFP-SpyTag-His BBa_K2273039 psB1C3-sfGFP-SpyCatcher-His BBa_K2273040 psB1C3-SpyTag-sfGFP-His BBa_K2273041 psB1C3-SpyCatcher-sfGFP-His BBa_K2273042

For the final construct we added the signal peptide sequence of amyE (BBa_K2273023) upstream of all constructs (Table 2) and cloned them into the single copy integrative B. subtilis vector pBS2EP_xylA. In brief, the vector has the following features for cloning in E.coli: an ori of replication and the bla gene mediating resistance against ampicillin. The B. subtilis specific part of the vector contains the multiple cloning site (MCS) in RFC10 standard with a P_xylA promoter upstream of the BioBrick prefix, a erm cassette providing resistance against erythromycin /lincomycin and flanking regions needed for integration into the lacA locus.

The sequenced plasmids were transformed into B. subtilis strain WB800N, a protease deficient strain. Fluorescence measurements of the supernatants derived from strains harbouring our constructs (Table 2) in the B. subtilis vector, was performed according to the protocol below.

SpyTag/SpyCatcher and FP (mCherry with C-terminal mini. SpyCatcher and C-terminal SpyTag, sfGFP with N-terminal SpyTag) were subcloned into the medium copy (15-20 copies per cell) B. subtilis vector pBS0E (Popp et al., 2017, accepted). Via Q5-PCR using the primers TM4067 and TM3082 and the corresponding pBS2E-plasmids as template. The PCR products were digested with BsaI und PstI and ligated with the EcoRI and PstI digested pBS0E vector backbone. The sequenced plasmids were transformed into B. subtilis strain WB800N. Fluorescence assay was performed as described above. Protein purification from the supernatants to check the functionality of the SpyTag/SpyCatcher and FP fusions was performed according to the protocol.

Fluorescence assay:

• Day cultures containing antibiotics in 2xYT medium were inoculated with the strains from an agarplate and grown for 8 h at 37°C
• 10 ml overnight culture with 1 % xylose in 2xYT medium were inoculated 1:50 from the day culture and grown for 16 h at 37°C
• To harvest the supernatant, 2 ml of the culture were centrifuged for 10 min at 3220 g
• The cell pellet was resuspended in fresh medium and tested as an control together with the supernatant and the cell culture
• The assay was implemented in a plate reader using a 96 well plate testing 100 μl of each sample
• The endpoint fluorescence of samples with sfGFP was measured at 510 nm with an excitation at 480 nm
• The excitation for mCherry was at 585 nm and the emission was measured at 615 nm

For all the assays biological duplicates and technical triplicates were used.

Purification and SDS-PAGE:

To prove the functionality of the SpyTag and the mini. SpyCatcher as a part of the fusion-proteins, the supernatants were purified with a quick protocol using agarose beads.

• Samples were mixed with a loading buffer containing a reducing agent and heated for 5 min at 95°C
• Then 10 μl were loaded onto a 12,5% SDS gel
• The gel was run at 200 V for 45 min. and stained in Coomassie Blue over night

References

[1]	Nijland, Reindert & Kuipers, Oscar. (2008). Optimization of Protein Secretion by Bacillus subtilis. Recent patents on biotechnology
[2]	Gilbert et. all (2017) Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS Synth. Biol.
[3]	Zakeri et. All (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Applied Microbiology and Biotechnology.
[4]	Li et. All (2013) Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag . J. Mol. Biol.
[5]	Overkamp, W. et al. Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. Environ. Microbiol. 79, 6481–6490 (2013).