Part:BBa_K5136223

I0500-B0034-LMT-GGG linker-T7 lysozyme 119G-SsrA-B0015

Biology

LMT

Building on the XMU-China 2022 (OMEGA) and 2023 (NAIADS) projects, we have further refined our research on signal peptides. In this year's study, we designed and compared the effects of eight different signal peptides, finding that the LMT signal peptide demonstrated superior performance in promoting protein excretion. Therefore, we have chosen to use LMT as the signal sequence for directing the secretion of proteins to the extracellular space or the cell membrane.

GGG linker

[(G4S)_n] is commonly used in protein engineering because of its flexibility and resistance to proteases. Therefore, we selected (GGGGS)₃ flexible linker (1) as a short peptide to connect LMT 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 (2, 3). 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 (3). 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 (4), and it differs from the T7 lysozyme 119V sequence selected from the UniProt database (5), with a variation at the 119^th 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 (6).

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 LMT, 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 (7).
This composite part we constructed aims to express the LMT-T7 lysozyme-SsrA mediated autolytic system (LLSA), which includes T7 lysozyme 119G, under the control of an L-arabinose inducible promoter. To validate the efficiency of the LLSA system, we used sfGFP as a reporter.

Figure 1 The expression gene circuits for the LLSA system.

Characterization

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136223) 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 (2278 bp) can be observed at the position around 3000 bp. (Figure 2).

Figure 2 Colony PCR of BBa_K5136223_pSB1C3 in E. coli DH10β. Target bands (2278bp) can be observed at the position between 2000 bp and 3000 bp.

sfGFP Release Efficiency Determination

After co-transforming I0500-B0034-LMT-GGG 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 LLSA system.

Figure 3 sfGFP release efficiency (%) (supernatant fluorescence intensity to bacterial culture fluorescence intensity) of the groups.

Refernce

1.D. Wen, S. F. Foley, X. L. Hronowski, S. Gu, W. Meier, Discovery and investigation of o-xylosylation in engineered proteins containing a (ggggs)n linker. Anal Chem 85, 4805-4812 (2013).
2.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). </br>3.F. W. Studier, Use of bacteriophage t7 lysozyme to improve an inducible t7 expression system. J Mol Biol 219, 37-44 (1991). </br>4.SnapGene.). Plyss. https://www.snapgene.com/plasmids/pet_and_duet_vectors_(novagen)/pLysS. </br>5.uniprot.). P00806 · enlys_bpt7. https://www.uniprot.org/uniprotkb/P00806/entry. </br>6.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). </br>7.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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961