Results

Construction

Our constructs were assembled with Golden Gate Assembly using the MoClo Toolkit from addgene.org. ^{generating...}

Level 0 parts refer to individual standardized parts such as a promoter, ribosome binding site, coding sequence or terminator.

Level 1 parts refer to transcriptional units. A transcriptional unit has a combination of parts needed to produce a protein, in it’s simplest model it may consist of a promoter, ribosome binding site, coding sequence, and terminator.

Modifications: We added SacI and XbaI restriction enzyme cut sites to the ends of our level 1 transcriptional units. The SacI site sits upstream of MWPp and XbaI sits downstream of our double terminator. With the new restriction enzyme sites, our final transcriptional units were used for assembly into a pSEVA3b67Rb backbone, making them compatible for use in Bacillus subtilis.

In this project, we assembled the following:

Level 0 Plasmids

Level 0 backbone: pAGM9121

• VP3
• CotB
• hyperfolder Yellow Fluorescent Protein (hfYFP)
• HisTag(8) 6-9
• HisTag(8) 6-7
• Rigid Linker 6-8
• Rigid Linker 7-8
• Thrombin-Cleavable Linker
• Photo-Cleavable Linker
• Middle Wall Promoter (MWPp)
• Double Terminator

Level 1 Plasmids

Level 1 backbone E.coli : pICH47732

Level 1 backbone B. subtilis : pSEVA3b67Rb

• MWPp-VP3-HisTag8(6-9)-Terminator (VH)
• MWPp-VP3-RigidLinker(6-8)-CotB-Terminator (VRC)
• MWPp-VP3-HisTag8(6-7)-ThrombinCleavable-CotB-Terminator (VHTC)
• MWPp-VP3-HisTag8(6-7)-PhotoCleavable-CotB-Terminator (VHPC)
• MWPp-VP3-hfYFP-RigidLinker(7-8)-CotB-Terminator (VYRC)
• MWPp-hfYFP-HisTag8(6-7)-ThrombinCleavable-CotB-Terminator (YHTC)
• MWPp-hfYFP-RigidLinker(7-8)-CotB-Terminator (YRC)

Validation

The agarose electrophoresis gels use NEB 1kb Plus as a DNA ladder placed on the leftmost side of the gels. DNA ladders do not count towards numbering. The level 1 composite parts are referred to by their abbreviations as listed in the “Level 1 Plasmids” section above.

Colony PCR Analysis - Lvl 0

Figure 1. Lanes: pAGM9121 (mini-prepped plasmid), pAGM9121 (single colony), VP3 (mini-prepped plasmid), Rigid Linker 7-8 (single colony), HisTag8 6-9 (single colony), HisTag8 6-7 (single colony), Double Terminator (single colony).

Figure 1. shows confirmation of VP3, Rigid Linker 7-8, His Tag(8) 6-9, His Tag(8) 6-7, and the Double Terminator.

Figure 2. Lanes: MWPp (mini-prepped plasmid), Photo-Cleavable (mini-prepped plasmid), Thrombin-Cleavable (mini-prepped plasmid), RigidLinker (mini-prepped plasmid), RigidLinker 6-8 (single colony), CotB (mini-prepped plasmid).

Figure 2. shows confirmation of MWPp, Rigid Linker 6-8 and CotB.

Colony PCR Analysis - Lvl 1

Figure 3. Lanes: MWPp-VP3-RigidLinker(6-8)-CotB-Terminator (VRC), MWPp-VP3-HisTag8(6-7)-ThrombinCleavable-CotB-Terminator (VHTC), MWPp-VP3-HisTag8(6-7)-PhotoCleavable-CotB-Terminator (VHPC), MWPp-VP3-HisTag8(6-9)-Terminator (VH) #1, MWPp-VP3-HisTag8(6-9)-Terminator (VH) #2

Figure 3. Shows first confirmation of MWPp-VP3-HisTag8(6-7)-ThrombinCleavable-CotB-Terminator (lane 2), MWPp-VP3-HisTag8(6-7)-PhotoCleavable-CotB-Terminator (lane 3), MWPp-VP3-HisTag8(6-9)-Terminator (VH) #1, and MWPp-VP3-HisTag8(6-9)-Terminator (VH) #2

Figure 4. Lanes: MWPp-VP3-HisTag8(6-7)-ThrombinCleavable-CotB-Terminator (VHTC), MWPp-VP3-HisTag8(6-7)-PhotoCleavable-CotB-Terminator (VHPC), MWPp-VP3-hfYFP-RigidLinker(7-8)-CotB-Terminator (VYRC), MWPp-hfYFP-RigidLinker(7-8)-CotB-Terminator (YRC)

Figure 4. shows confirmation of MWPp-VP3-HisTag8(6-7)-ThrombinCleavable-CotB-Terminator (lane 1) and MWPp-VP3-HisTag8(6-7)-PhotoCleavable-CotB-Terminator (lane 2).

Summary

Confirmed Level 0’s:

• VP3
• CotB
• hyperfolder Yellow Fluorescent Protein (hfYFP)
• HisTag(8) 6-9
• HisTag(8) 6-7
• Rigid Linker 6-8
• Rigid Linker 7-8
• Thrombin-Cleavable Linker
• Photo-Cleavable Linker
• Middle Wall Promoter (MWPp)
• Double Terminator

Unconfirmed Level 0’s:

• All confirmed

Confirmed Level 1’s:

• MWPp-VP3-HisTag8(6-9)-Terminator (VH)
• MWPp-VP3-HisTag8(6-7)-ThrombinCleavable-CotB-Terminator (VHTC)
• MWPp-VP3-HisTag8(6-7)-PhotoCleavable-CotB-Terminator (VHPC)

Unconfirmed Level 1’s:

• MWPp-VP3-RigidLinker(6-8)-CotB-Terminator (VRC)
• MWPp-VP3-hfYFP-RigidLinker(7-8)-CotB-Terminator (VYRC)
• MWPp-hfYFP-RigidLinker(7-8)-CotB-Terminator (YRC)

“Heat Innactivation”:

One of our initial ideas was to heat innactivate bacteria for its final use. But our concern is that this may destroy or alter the native conformation of VP3 (or any other displayed protein). We also decided to see if our fluorescent cells will be of use for in vivo tests in bee tissues after autoclaving. We found out that this would hinder the RFPs ability to fluoresce and therefore not allow us to visualize our cells with microscopy. This is one reason why we would do tests with leaving modified cells and bees in the future for localization purposes.

E. coli containing pseva3b67rb plasmids (left autoclaved, right non-autoclaved).

In Progress

Verification is still ongoing to confirm the external cell surface display of our recombinant proteins through microscopy and cleavable linkers.

Microscopy:

Fluorescent cells: Cells which display the hyper folder yellow fluorescent protein on the surface of the cell surface are being used for fluorescent microscopy.

Figure 5. E. coli DH5a with cytosolic RFP

Figure 5. shows our first attempt of visualizing our fluorescent cells.

Fluorescence in bee tissues: The machine (vibratome) required for slicing honey bee tissue with our fluorescent cells is currently broken. Control samples of some of our cells were obtained with a confocal microscope. We are continuing to gather samples for microscopy, however, imaging is delayed due to the broken vibratome.

Cleavable Linkers:

Current efforts of transformation are underway in E. coli DH5a and B. subtilis WB800N with the level 1 constructs. If the recombinant proteins consisting of VP3-PhotoCleavable-CotB and VP3-ThrombinCleavable-CotB are successfully brought to the cell surface, then they can be verified by cleaving the linkers, either by wavelength or a thrombin proteases. The cleaved protein in the lysate will be verified with an SDS-PAGE gel.

Future Plans

###Molecular:

1. Implement different methods of protein surface display to optimize the most robust VP3 display in our cells.
2. Implement a backbone plasmid that is unable to be taken up by natural transformation so that if the constructs were used by beekeepers, there would be no possibility of other microbes taking up our constructed plasmid.
3. Or, create chromosomal integration into Bacillus subtilis for all of our constructs.
4. Evaluate different methods of Bacillus subtilis deactivation to see which methods best reserve the folding of our surface proteins while fully deactivating the bacteria.
5. Perform further analysis to determine the correct folding of our VP3 protein after transport to the cell surface.

###Microscopy:

1. Imaging with additional resources such as dyes for actin filaments to better illustrate the dimensions of the bee cells. Dedicate fundraising for the purchase of primary antibodies for our heterologous surface proteins to more accurately show their presence in our bee tissues.

###DWV tests:

1. With support from Abbi Chapman, from the University of British Columbia (Vancouver), we will be sending our deactivated B. subtilis cells with our recombinant protein attached to the cell surface.

By the end of this October, if our constructs are working correctly, Abbi will be conduct a feeding experiment with the vaccine. She will then infect the honey bees with Deformed Wing Virus (DWV) to see if the initial vaccination decreases the amount of DWV titers in the treated bees.

2. Evaluate the upregulation of immune genes after the feeding of our vaccine and during infection of DWV, and compare with non immunized gene regulation.

###Next year:

1. Further expansion of this project is likely to continue in the next iGEM season.

2. Further expansion of this project is likely to continue in the next iGEM season.