Part:BBa_K2812005

Coding sequence for trunctated Lysostaphin with HlyA and His6-tag regulated by T7-promoter

The biobrick contains the coding domain for truncated lysostaphin, based on the coding sequence derived from BBa_K748002. This was fused to the C-terminal sequence of Hemolysin A (HlyA) via a thrombin linker to allow secretion of the lysostaphin. In this biobrick, lysostaphin-HlyA production is regulated by the T7 promoter BBa_K525998. Expression can be induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG). TU-Eindhoven 2018 used this part to secrete lysostaphin from Escherichia coli to kill Staphylococcus aureus biofilms for the treatment of wound infections. For more information about our project, please visit our [http://2018.igem.org/Team:TU-Eindhoven wiki].

Usage & Biology

Lysostaphin

Lysostaphin is an antimicrobial agent produced by Staphylococcus simulans. It targets the cell wall peptidoglycan found in certain Staphylococci by cleaving its cross-linking pentaglycine bridges. Among others, it is effective for degrading Staphylocuccus aureus biofilms.¹ The encoding part of the lysostaphin has been derived from BBa_K748002, which was made by iGEM Harbin 2012 and was also used by iGEM Stockholm 2016. iGEM Eindhoven 2018 codon optimized this lysostaphin construct. Lysostaphin belongs to the major class of antimicrobial proteins and peptides known as bacteriocins. Bacteriocins are proteins or peptides produced by bacteria, displaying a bactericidal activity against other subpopulations of bacteria.² The cell wall degradation capability of lysostaphin derives from its endopeptidase activity on pentaglycine cross-bridges in the peptidoglycan layer. Specific cleavage between the third and fourth glycine residue leads to the destruction of the peptidoglycan layer and subsequent lysis of the bacteria.

HlyA

The C-terminal sequence of Hemolysin A (residues 807-1024 of the E. coli HlyA gene) functions as a non-cleavable signal peptide for protein translocation via the Type I secretion pathway of Gram-negative bacteria. Here, HlyA is fused to the lysostaphin for its secretion from E. coli BL21 (DE3) cells. In Type I secretion, single step transport of the target protein occurs from the cytoplasm to the extracellular environment. The HlyA is fused to the C-terminus of the target protein.³ It is a 23-kDa signal sequence that targets proteins for secretion via Type I secretion pathway. HlyA is secreted into the medium in a TolC and HlyB/D-dependent manner, in the presence of CaCl₂.⁴ It is recognized by the membrane translocation complex composed of HlyB and HlyD, which together with the TolC protein, will form a pore through the membrane.⁵ This will lead to the secretion of HlyA-containing fusion proteins. Figure 1 illustrates the steps involved in the type I secretion of lysostaphin as an HlyA fusion protein.

T7 promotor, Thrombin linker & His-tag

To control protein expression of lysostaphin-HlyA, a T7 promotor has been assembled to the fusion protein. The T7 promotor can be regulated by IPTG. The lysostaphin and HlyA are linked via a thrombin linker. It is a short peptide sequence which can be cleaved by the enzyme thrombin, resulting in the removal of the HlyA domain. This avoids interference with the functionality of lysostaphin. C-terminal to the HlyA domain, a His-tag (6x repeated amino acid histidine) is attached. The His-tag can be used for detection of the fusion protein by e.g. Western Blot, but also for protein purification purposes as it facilitates binding to a nickel affinity column.

Fusion protein

The combination of all components described above, lead to a construct that can be (continuously) secreted by E. coli if the HlyB/D coding sequences have been co-transformed. Running the medium containing the fusion protein over a nickel affinity column and subsequent thrombin linker cleavage allows for the easy isolation of purified truncated lysostaphin.

Figure 1: Type I secretion of lysostaphin via HlyA, HlyB, HlyD and TolC. IM = inner membrane; OM = outer membrane; A = HlyA; L = Lysostaphin

Experimental Characterisation by TU-Eindhoven (2018)

Figure 1: 1% agarose gel of the colony PCR of the T7-Lysostaphin-HlyA biobrick.

Cloning

TU-Eindhoven 2018 has characterized the biobrick BBa_K2812005 at both the DNA and the protein level. First, the lysostaphin-thrombin linker-HlyA-His-tag construct BBa_K2812004 was synthesized by IDT and assembled behind the T7 promotor of iGEM Bielefeld 2011 BBa_K525998 using restriction digestion by EcoRI and PstI followed by ligation. Subsequently, the contruct was double digested and assembled into the digested linearized pSB1C3 backbone via ligation. The ligated construct was successfully transformed into E. coli NovaBlue, followed by a colony PCR using the VF2 and VR primers to investigate if the correct length has been inserted in the vector. The mixture was ran on a 1% agarose gel as can be seen in figure 1. The observed length of the brightest band corresponds with the expected length of 1810 basepairs, confirming that the desired construct has been succesfully ligated in pSB1C3 and subsequently transformed in E. coli NovaBlue. Next, the colonies with the correct insert were cultured in LB medium before a plasmid purification by a miniprep. The isolated plasmid DNA was sent for Sanger sequencing and the sequence could be confirmed.

Protein Expression

Figure 2: SDS-PAGE gel of the T7-Lysostaphin-HlyA biobrick protein expression experiment.

After the successful characterisation of the biobrick at the DNA level, protein expression experiments were performed. The biobrick was successfully transformed into BL21 (DE3), after which a culture was set up at 37 °C. A sample was taken prior to induction (t0) to establish gene expression pattern of the uninduced bacteria. The cultures were induced at an OD600 of 0.5-0.8 by adding 0.5 mM IPTG to induce expression of the recombinant protein, also at 37 °C. Samples were taken 3 hours (3h) after induction and after overnight incubation (o/n). SDS samples were prepared and loaded onto a polyacrylamide gel to yield the SDS-PAGE results that can be seen in figure 2.

The expected length of the biobrick is 50 kDa. In the uninduced sample (t0), no bands indicative of overexpression of (any) protein can be observed. 3 hours after induction (3h) of protein expression, two bands indicating overexpression can be seen; one at 50 kDa and one at 25 kDa. After overnight induction, the band at 50 kDa has disappeared completely and the band around 25 kDa has increased in intensity. The SDS sample of 3h indicates successful induction of expression of the construct. The band at 25 kDa is not expected. The disappearance of the band at 50 kDa and increase of the 25 kDa band can be explained by cleavage of the thrombin linker due to accumulation of lysostaphin in the cytosol. The thrombin linker used in the design contains two times a GGGGS repeat. The substrate sequence of lysostaphin is GGGGG. However, it is known that the bacterial species S. simulans incorporates serine residues at the third and fifth position in the cell wall cross bridges (GGSGS), resulting in a 1000-fold decrease in susceptibility to lysostaphin as lysostaphin cannot hydrolyse glycylserine and serylglycine bonds.¹ Lysostaphin is thus still able to cleave these linkers, although at a lower rate. If the thrombin linker between lysostaphin and HlyA is cleaved by another lysostaphin enzyme, it yields two proteins of 27 kDa (lysostaphin) and 23 kDa (HlyA). The band around 25 kDa and the disappearance of the 50 kDa band can thus be explained by lysostaphin cleaving the thrombin linker. From this experiment it can be concluded that our construct can be induced successfully.

Sources

1) Tossavainen, H., Raulinaitis, V., Kauppinen, L., Pentikäinen, U., Maaheimo, H., & Permi, P. (2018). Structural and Functional Insights Into Lysostaphin–Substrate Interaction. Front Mol Biosci.

2) Bastos, M. d., Coutinho, B. G., & Coelho, M. L. (2010). Lysostaphin: A Staphylococcal Bacteriolysin with Potential Clinical Applications. Pharmaceuticals (Basel), 1139–1161.

3) Thomas, S., Holland, I. B., & Schmitt, L. (2014). The Type 1 secretion pathway — The hemolysin system and beyond. Molecular Cell Research, 1629-1641.

4) Gray, L., Baker, K., Kenny, B., Mackman, N., Haigh, R., & Holland, I. (1989). A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium. J Cell Sci Suppl., 45-57.

5) Wandersman, C., & Delepelaire, P. (1990 ). TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A., 4776-4780.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1389
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 164
  
• 1000
  COMPATIBLE WITH RFC[1000]