Part:BBa_K4733008

Spore Coat Protein B

Spore coat protein B (CotB) is a protein found in the spore coat of the gram-positive B. Subtilis. CotB contains an anchoring motif that allows it to attach to the cell’s outer membrane. CotB is 59 kDa in size and has a highly hydrophilic C-terminus formed by amino acid repeats containing, glutamine, lysine, and serine residues (Ricca et al. 2003)

It has been known that when a protein of interest is fused to the C-terminus or N-terminus of B. Subtilis anchor protein (i.e. CotB, CotB, CotC, CotG, CotZ, etc), the fusion protein will be displayed on the surface of B. Subtilis spores (Zhang et al. 2020). However, Han et al. (2020) showed that a recombinant protein fused to CotB can also be exported to the outer membrane of vegetative B. Subtilis cells. These researchers used this innovative display system to express on the surface of B. Subtilis cells the foreign fusion protein: immunodominant ovalbumin T-cell epitope fused with the cholera toxin B subunit. They fused this recombinant protein to the C-terminus of CotB and confirmed it was successfully exported to the extracellular membrane through flow cytometry, immunofluorescence microscopy, and western blotting.

CotB’s sequence was modified by the 2023 UBC-Okanagan team so that it does not contain any illegal restriction type II sites (BbsI and BsaI) since we followed Modular Cloning (MoClo) standards to assemble our constructs.Additionally, a stop codon was added at the end of the sequence.

Advantages and Disadvantages

The main advantage of CotB is its flexibility and ability to display foreign proteins on the outer membrane of B. subtilis. Additionally, Yuan et al. (2020) state that CotB belongs to B. subtilis a not pathogenic species meaning it will not be harmful to hosts in case of use as a drug or vaccine delivery system. So much so that Bacillus subtilis 168 was modified to produce Natto (fermented soybeans).

That being said, the heterologous proteins expressed on the surface of B. Subtilis have been shown to degrade significantly over time. Furthermore, Han et al. (2011) stated that the amount of heterologous protein expressed by this display system is not very high, and as such more work needs to be done to increase the yield.

Usage and Biology

This is due to this part containing an AATG 5' flank which does not match the correct amino acid reading frame which should start on the next ATG sequence.

How did we use it? The 2023 UBC-Okanagan team used this part for a vaccine delivery system. The system involves fusing CotB to a capsid protein, VP3, belonging to the Deformed Wing Virus. A linker was used to maintain the integrity of both of these proteins. To the lvl 0 CotB part, the team added the MoClo flanks 8 and 9 to the N and C terminus of the protein (refer to figure 1) so that during the assembly of parts, CotB would be on the C-terminus of the fusion protein. The idea behind this system is to have VP3 function as an antigen displayed on the outer surface of B. Subtilis, which will be ingested by a Bee: thus eliciting a favorable immune response in the bee, developing an immunity.

[Figure 1. CotB construct]]

How is it useful to the community? As stated earlier, CotB allows the display of recombinant proteins on the surface of live bacterial cells, which is extremely beneficial for the development of drug and enzyme production systems, other vaccine delivery systems, and even control of environmental pollution through its use as a bioremediation tool for adsorbing heavy metal ions according to Krzysztof et al. (2010).

Modelling

Using protein sequence and Alphafold we modeled the shape and structure of CotB and visualized it using ChimeraX (Figure 2). Moreover, using Orientations of proteins in membranes database and software (OPM) by The University of Michigan we generated a rough estimate of how CotB would look on the surface of a gram-positive bacteria (figure 3).

[Figure 2. CotB modeling]]

[Figure 3. CotB displayed on the surface of Gram-Positive Bacteria]]

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 911
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 198