Part:BBa_K5136030

_NOTOC__ FhuD-TSS linker-T7 lysozyme 119G-SsrA

Biology

FhuD

In Escherichia coli, protein translocation guided by signal peptides primarily employs two distinct mechanisms: the Sec pathway and the Tat pathway. Notably, some proteins are capable of utilizing both pathways for their translocation (1) . The FhuD signal peptide, acting as an intrinsic dual Sec-Tat pathway (2), is frequently employed in biotechnological applications to direct the secretion of proteins to the extracellular space or the cell membrane. This characteristic makes the FhuD signal peptide an ideal choice for constructing secretion expression vectors, particularly in applications aimed at enhancing the yield of target proteins.

TSS linker

We identified a linker used for constructing surface-display fusion proteins from previous research projects, which has demonstrated certain advantages in the assembly of fusion proteins. Based on this, we attempted to use the TSS linker sequence（Parts | XMU-iGEM 2022，Team:XMU-China/Parts - 2020.igem.org）, which has been validated in the surface display system, as a short peptide to connect FhuD and T7 lysozyme 119G in our autolytic system.

T7 lysozyme 119G

T7 lysozyme is a small molecular weight protein in bacteriophage T7, primarily functioning to degrade the cell wall of host bacteria during phage infection, facilitating the injection of phage DNA or the release of newly formed phage particles. In molecular biology research, it is widely used for the efficient lysis of Escherichia coli cells (3, 4). Moreover, it has been reported that higher levels of lysozyme provided by plasmids pLysE or pLysH can reduce the full induction activity of T7 RNA polymerase, allowing induced cells to continue growing indefinitely while producing non-toxic target proteins (4). This feature not only highlights the excellence of T7 lysozyme in promoting cell lysis but also makes it extremely useful in preparing cell extracts for protein purification. Notably, T7 lysozyme 119G sequence was found in pLysS (5), and it differs from the T7 lysozyme 119V sequence selected from the UniProt database (6), with a variation at the 119th amino acid position.

SsrA

The SsrA is a small peptide tag used to mark proteins for protein degradation. When fused with the target protein, SsrA could guide it to specific proteases, such as the ClpXP and ClpAP complexes, for degradation (7).

Usage and Design

In our design, we aim to induce cell autolysis to release enzymes into the supernatant, simplifying the complex protein purification process. By utilizing the dual-pathway signal peptide FhuD, we direct T7 lysozyme to the peptidoglycan layer, enhancing cell lysis. Additionally, the SsrA tag is fused to the C-terminus of T7 lysozyme to ensure the degradation of any leaked T7 lysozyme, minimizing system cytotoxicity and ensuring the proper accumulation of the target enzyme in the correct location (8). To construct our composite part, we utilized the promoter (BBa_I0500), RBS (BBa_B0034), FhuD-TSS linker-T7 lysozyme 119G-SsrA coding sequence (BBa_K5136030), and terminator (BBa_B0015). This composite part we constructed aims to express the FhuD-T7 lysozyme-SsrA mediated autolytic system (FLSA), which includes T7 lysozyme 119G, under the control of an L-arabinose inducible promoter. To validate the efficiency of the FLSA system, we used sfGFP as a reporter.

The expression gene circuits for the FLSA system.

Characterization

Agarose Gel Electrophoresis (AGE)

The composite part (BBa_K5136220) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli DH10β. The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (2335 bp) can be observed at the position around 3000 bp. (Figure 2).

Figure 2 Colony PCR of BBa_K5136220_pSB1C3 in E. coli DH10β. Target bands (2335bp) can be observed at the position between 2000 bp and 3000 bp.

sfGFP Release Efficiency Determination

After co-transforming I0500-B0034-FhuD-TSS linker-T7 lysozyme 119G-SsrA-B0015_pSB1C3 and sfGFP_pET-28a(+) into E. coli BL21 (DE3), the cultures were grown overnight in LB medium containing corresponding antibiotics. The cultures were diluted and grown to OD₆₀₀ 0.6-0.8, followed by the addition of 0.5 mM IPTG to induce sfGFP expression at 18°C. After 10 hours, 0.25% L-arabinose was added to activate the autolytic system. The total fluorescence intensity was measured after 16 hours of expression of the induced autolysis system, and after centrifugation, the fluorescence intensity of the supernatant was measured too. The ratio of the fluorescence intensity of the culture and supernatant was used to assess the lysis efficiency of FLSA system.
By comparing with the control group (Figure 3), we determined that the FLSA system (FhuD-TSS linker-T7 lysozyme 119G-SsrA) which harboring TSS linker between FhuD signal peptide and T7 lysozyme had an sfGFP release efficiency of ~22%. In addition, from Figure 3, we also found the effect of different linkers and T7 lysozyme of different versions to the sfGFP release efficiency, which emphasizes the significance of linker sequence between different functional elements and the priciseness of amino acid sequence.

Figure 3 sfGFP release efficiency (%) (supernatant fluorescence intensity to bacterial culture fluorescence intensity) of the groups. Lysis efficiency of the dual-plasmid transformants harboring I0500_pSB1C3 and sfgfp_pET-28a(+) (negative control, corresponding to ①) and the dual-plasmid transformants harboring I0500-B0034-FhuD-TSS linker-T7 lysozyme 119G-SsrA-B0015_pSB1C3 and sfgfp_pET-28a(+) (Experimental group, corresponding to ④)after 16 hours of induction. p-value: <0.0001 (****).

Reference

[1] D. Tullman-Ercek et al., Export pathway selectivity of escherichia coli twin arginine translocation signal peptides. J Biol Chem 282, 8309-8316 (2007).
[2] F. Zhang et al., N-terminal fused signal peptide prompted extracellular production of a bacillus-derived alkaline and thermo stable xylanase in e. Coli through cell autolysis. Appl Biochem Biotechnol 192, 339-352 (2020).
[3] J. Yun, J. Park, N. Park, S. Kang, S. Ryu, Development of a novel vector system for programmed cell lysis in escherichia coli. J Microbiol Biotechnol 17, 1162-1168 (2007).
[4] F. W. Studier, Use of bacteriophage t7 lysozyme to improve an inducible t7 expression system. J Mol Biol 219, 37-44 (1991).
[5] SnapGene.). Plyss. https://www.snapgene.com/plasmids/pet_and_duet_vectors_(novagen)/pLysS.
[6] uniprot.). P00806 · enlys_bpt7. https://www.uniprot.org/uniprotkb/P00806/entry.
[7] Q. Chai, Z. Wang, S. R. Webb, R. E. Dutch, Y. Wei, The ssra-tag facilitated degradation of an integral membrane protein. Biochemistry 55, 2301-2304 (2016).
[8] F. Zhang et al., Development of a bacterial fhud-lysozyme-ssra mediated autolytic (flsa) system for effective release of intracellular products. ACS Synth Biol 12, 196-202 (2023).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]