Coding

Part:BBa_K3416029

Designed by: Eglė Vitkūnaitė   Group: iGEM20_Vilnius-Lithuania   (2020-10-26)
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gp529

Introduction

FlavoFlow

Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This part was used for the second goal- treatment - of the project FlavoFlow.

Biology

Bacteriophages or any other virus can inject its genomic material into the host cell environment. But to achieve it virus needs to create a passage throughout diverse carbohydrate barriers such as O-polysaccharide chains of lipopolysaccharide molecules, capsule polysaccharides, peptidoglycan layers, or even extracellular polysaccharides which can form biofilm matrix1. That is why viruses have a big arsenal of proteins, mostly located on its tail spikes, which can penetrate this polysaccharide obstacle. Sometimes, the amount of these proteins is the only factor that defines bacteriophage’s host spectrum and specificity2. Exolysins are also called depolymerases which are a component of the adsorption apparatus, most commonly with two domains essential for viral infection. The first domain responsible for initiating binding to the capsule and the second one is responsible for digestion of the capsule. Biochemically speaking, depolymerases could be one of two groups - lyases or hydrolases. The biggest difference between them is that when lyase cleaves a specific bond of a substrate, it does not consume water molecules from the environment. That could be a significant advantage if the enzyme should catalyze the reaction in a waterless environment. Exolysins gp529 (BBa_K3416029), gp531 (BBa_K3416031), gp533 (BBa_K3416033) belong to the giant singleton phage RAK-2 which infects multidrug-resistant Klebsiella pneumoniae KV-3 2020 Vilnius-Lithuania iGEM teamused synthetic biology to design a construct of an AI-2 induced exolysin-endolysin system.

Results

To verify that our proteins had exolysin activity we had to do bioinformatic analysis. By comparing the sequences of the genes and their gene products, we can predict that they are tail fiber proteins. Also, by using the Basic Local Alignment Search Tool we have found that these proteins show high similarities(>85%) with K. pneumoniae phage K64-1 tail spike proteins which have been proven responsible for depolymerase of the bacteria polysaccharides4. Also, gp533 (BBa_K3416033) is homologous (85,58%) to the gene which will result in a protein with pectate lyase enzymatic activity. After exolysin sequence analysis, we can deduct that our proteins have pectate lyase activity. Pectate lyase is responsible for eliminating cleavage of pectate, yielding oligosaccharides with 4-deoxy-α-D-mann-4-enuronosyl at their non-reducing ends (Fig. 1).

Figure 1.Pectate lyase catalyzed reaction

2020 Vilnius-Lithuania iGEM team presents gene constructs where exolysin gp533 (BBa_K3416033) is incorporated into genetic circuit under quorum sensing promoter which is inducible with phosphorylated autoinducer-2.

To test exolysins activity, we tried to induce and purify three different exolysins (BBa_K3416029), [(https://parts.igem.org/Part:BBa_K3416031 BBa_K3416031),] [(https://parts.igem.org/Part:BBa_K3416033 BBa_K3416033).] Plasmids with the cloned exolysins were kindly provided by prof. Rolandas Meškys.

Plasmid pET28b(+)-gp529 was transformed into Rosetta (DE3) strain and induced with 0.5mM IPTG for 19 hours in 16°C in 200mL LB. Biomass was resuspended into 50mM Tris-HCl ph=8 and ultrasound lysed until the solution became clear. His-tagged protein was purified using Ni-NTA. Dialysed and concentrated. (Fig. 2) Yielding a 1.2mg of the protein from 200mL of LB medium. But the protein was not in its active form. Thus, more optimization is required (Fig. 3,4).

After purification, we tested exolysin activity using the spot test on Klebsiella pneumonia KV-3 strain (Fig. 3,4). First, we performed an experiment to determine a minimal amount of protein needed to form a lysis halo (Fig.3). 5μL of 4μM initial (101) starting concentration of all exolysins were used. Dilutions were 101, 10-1, 10-2, 10-3, 10-4, 10-5. After spot test analysis it was seen the exolysin gp529 did not show any depolymerase activity towards this strain, while gp531 had the most visible activity. After dilution experiments, the smallest measured exolysin concentration, which can induce cell lysis is 10-2 – 10-3 of initial concentration. However, even if this part works, after sequencing we saw that this gene sequence is not compatible with iGEM standard construction. Due to this, we decided not to use it in our genetic circuit. However, nonetheless, gp533 showed minimal but detectable depolymerase activity so we decided to construct it under AI-2 induced promoter. For the spot test positive control we have chosen RAK-2 phage, which was kindly provided by doc. Algirdas Noreika. Phage dilution was 101, 10-2, 10-4, 10-6, 10-8, 10-10. As the negative control, we used 50 mM Tris–HCl pH=8.

We also tested the temperature sensitivity of the enzymes. 24μmol of exolysins were incubated for 5 minutes at 42oC, 60oC, 65oC, 70oC. Spot assay confirms that exolysins have temperature resistance as gp531 could withstand 65°C and gp533 could withstand 60°C (Fig. 4). The most distinct halos for gp531 and for gp533 were at 60°C.

  • Figure 2.SDS-PAGE electrophoresis results after gp529, gp531, gp533 purification. L - PageRulerTM Prestained Protein Ladder (#SM0671) (Fermentas), 1 – gp529 biomass sample before induction, 2 – gp529 biomass sample after induction, 3 -purified and highly concentrated gp529 (62.5 kDa), 4 - gp531 biomass sample before induction, 5 – gp531 biomass sample after induction, 6 - gp531 purified protein (97.8 kDa), 7 - gp533 biomass sample before induction, 8 - gp533 biomass sample after induction, gp533 purified protein (82.2 kDa).
  • Figure 3. Spot test with serial dilutions of gp529, gp531, gp533 exolysins. From top to bottom the proteins were gp529, gp531, gp533, RAK-2(positive control), blank(negative control). Tris-HCl was used as a negative control. From left to right the protein dilutions were 101, 10-1, 10-2, 10-3, 10-4, 10-5 and phage dilutions were 101, 10-2, 10-4, 10-6, 10-8, 10-10.
  • Figure 4. Spot test with gp529, gp531, gp533 exolysins (24μmol) incubated at 42°C, 60°C, 65°C, 70°C . From top to bottom the proteins were gp529, gp531, gp533, RAK-2(positive control), blank(negative control). RAK-2 phage (1.2 × 1010 PFU/mL) was used as a positive control. Tris-HCl was used as a negative control.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 736
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 736
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 736
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 736
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1514


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