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| | To evaluate the efficiency of the secretion process we monitored the fluorescence of B. subtilis strains carrying our constructs and compared them to the wild type. In order to prove the functionality of the SpyTag/SpyCatcher system we performed an SDS-Page demonstrating the formation of a fusion protein derived from co-incubated supernatants. </p> | | To evaluate the efficiency of the secretion process we monitored the fluorescence of B. subtilis strains carrying our constructs and compared them to the wild type. In order to prove the functionality of the SpyTag/SpyCatcher system we performed an SDS-Page demonstrating the formation of a fusion protein derived from co-incubated supernatants. </p> |
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| − | <div class="contentbox">
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| − | <h1 class="box-heading">Design</h1>
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| − | <p>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.
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| − | The gene encoding the mini. SpyCatcher (BBa_K2273015) was chemically synthesized.
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| − | The codon optimized SpyTag (BBa_K2273014) was generated via overlapping primers iG17P049 and G17P050 and amplified using the primers TM4487 and iG17P039 (LINK). 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).
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| − | The His-tag, necessary for protein purification was included in the reverse primers (table 1).
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| − | Table 1: Overview of the constructed basic parts.
| + | <div class="contentbox"> |
| − | Gene BioBrick No. forward Primer Reverse Primer
| + | <h1 class="box-heading">References</h1> |
| − | SpyTag_His-tag BBa_K2273016 TM4487 iG17P068
| + | <table class="source"> |
| − | Mini. SpyCatcher_His-Tag BBa_K2273017 TM4487 iG17P067
| + | <colgroup> |
| − | sfGFP_His-tag BBa_K2273021 TM4487 iG17P065
| + | <col width="5%"> |
| − | mCherry_His-tag BBa_K2273022 TM4487 iG17P066
| + | <col width="95%"> |
| − | | + | </colgroup> |
| − | | + | <tr> |
| − | In order to identify the best combination of SpyTag/SpyCatcher and FP fusion, we constructed all N- and C-terminal combinations (table 2).
| + | <td><a target="_blank" href ="https://www.researchgate.net/profile/Reindert_Nijland/publication/23656731_Optimization_of_Protein_Secretion_by_Bacillus_subtilis/links/09e415136142e1554c000000/Optimization-of-Protein-Secretion-by-Bacillus-subtilis.pdf">[1]</a></td> |
| − | | + | <td>Nijland, Reindert & Kuipers, Oscar. (2008). Optimization of Protein Secretion by <i>,Bacillus subtilis</i>,. <i>,Recent patents on biotechnology</i></td> |
| − | Table 2: List of SpyTag/SpyCatcher and FP composite parts.
| + | </tr> |
| − | pSB1C3-mCherry-SpyTag-His BBa_K2273035
| + | </table> |
| − | pSB1C3-mCherry-SpyCatcher-His BBa_K2273036
| + | </div> |
| − | 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 pBS2EPxylA. 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 PxylA promoter upstream of the BioBrick prefix, a erm cassette providing resistance against erythromycin / lincomycin and flanking regions needed for integration into the lacA locus.
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| − |
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| − | 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 attached protocol (LINK).
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| − | Image 1- 8
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| − | After the initial screening, the three most promising combinations of 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 (LINK).
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| − |
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| − | Fluorescence assay:
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| − | 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 l 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.
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| − | For all the assays biological duplicates and technical triplicates were used.
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| − |
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| − | Purification and SDS-PAGE:
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| − | 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 (Link).
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| − | The samples were mixed with a loading buffer containing a reducing agent, heated for 5 min at 95°C and then 10 l 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.
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| − | </p>
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| − | </div>
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| | </p> | | </p> |
| | </div> | | </div> |
