Parts Overview
Plasmid
1. We used plasmidbackbone pSTK-0-sfGFP to clone our level 0 DNA construct and prepare for thenext-level construct. We obtained it from STK toolkit. The plasmid contains thefollowing components:
- Multiple cloning site (MCS) flanking with BsaI and BsmBI sites
- One selection marker:
- A chloramphenicol (Cm+) resistant gene for selection of successfully transformed E. coli cells
- Marker for selections: superfolding green fluorescentprotein(sfGFP), enables us to identify the insertion of target sequences
- Marker for selections: superfolding green fluorescentprotein(sfGFP), enables us to identify the insertion of target sequences
- sfGFP ribosome binding site (RBS)
- Origin of replication
Figure 1: Scheme of a Pstk-0-sfGFP backbone.
2. We used plasmid backbone STK002_ pSTK-1A-sfGFP, STK002_ pSTK-1B-sfGFP, STK002_ pSTK-1C-sfGFP, and STK002_ pSTK-1D-sfGFP to clone our level 1 DNA construct and prepare for the next-level construct. We obtained it from the STK toolkit. The plasmids contain the following components:
- Multiple cloning site (MCS)
- One selection marker:
- A bla coding sequence (CDS), which provides resistance to the antibiotic ampicillin (Amp+).
- Marker for selections: superfolding green fluorescentprotein(sfGFP), enables us to identify the insertion of target sequences
- sfGFP ribosome binding site (RBS)
- ColE1 origin
Figure 2: Scheme of a STK002_pSTK_1A-sfGFP backbone.
Figure 3: Scheme of a plasmid inserted with gRNA.
3. We used pSTK-1A-lacZ, pSTK-1B-lacZ, and pSTK-1C-lacZ as backbones for inserts with GPFin them. The plasmids contain the following components:
- Multiple cloning site (MCS)
- One selection marker:
- A bla coding sequence (CDS), which provides resistance to the antibiotic ampicillin (Amp+).
- A lacZ reporter cassette, in which successful integration of the target sequence will inhibit the express of lacZ gene.
- Marker for selections: superfolding green fluorescent protein (sfGFP), enables us to identify the insertion of target sequences
Figure 4: Scheme of a pSTK-1A-lacZ backbone with insertedCotG, the flexible linker, and chitinase.
4. We used STK-EXP-1-sfGFPas a backbone to express proteins in B. subtilis. The target gene was inserted in the vector, which could allow rapid, nontoxic selection of successfully transduced cells. We obtained it from STK toolkit. The plasmid contains the following components:
- MCS flanking with BsaI and BsmBI sites
- One selection marker:
- A chloramphenicol (Cm+) resistant gene forselection of successfully transformed B. subtilis cells.
- Marker for selections: superfolding green fluorescent protein (sfGFP), enables us to identify the insertion of target sequences
- sfGFP ribosome binding site (RBS)
- ColE1 origin
Figure 5: Scheme of STK-EXP-1-sfGFP inserted with CotZ and chitinase.
Chitinase Display
In order to display the chitinase on the surface of B. subtilis, we assembled chitinase with 2 coat proteins of B. subtilis spores, CotGand CotZ. In this case, we are able to display chitinase on the surface of B. subtilis spores. The part BBa_K4212034 contains CotG with chitinase, and the part BBa_K4212038 (contains CotZ with chitinase) .
We also added flexible linkers to the assembly since based on our drylab models, the addition of linkers for the surface protein CotG can reduce steric hindrance between the coat proteins with chitinase. We intended to verify the models so we employed different linkers. The part BBa_K4212035 is with the linker 3Gx3; BBa_K4212036 is with 4Gx3; and BBa_K4212037 is with AHx3.
Self-Digesting Plasmid
Our self-digesting plasmid is consisted of 3 parts: gRNA, D15, Cas9 and GFP_mut3b .Each subparts include multiple elements:
- Part BBa_K4212044 has PlepA promoter, RBS2, gRNA, and Spy terminator.
- Part BBa_K4212045 has PsspB promoter, RBS1, D15, and 0D spacer.
- Part BBa_K4212046 has 0A spacer, optimal RBS, Cas9, and B0015terminator
- Part BBa_K4212047 has hyperspank promoter, RBS3, GFP_mut3b, tL3S2P21terminator.
For more working mechanism and design about each construct, please seeour design page (should link to design page)
Germinant Receptor Engineering
We focused on the germinant receptor GerA, which in wild-type B. subtilisrecognises L-alanine. As a novel approach, we planned to rewire GerA, sothat it has specificity towards N-acetylglucosamine (NAG) - the chitin monomersinstead. In order to achieve that, we designed GerA-GerK chimera, which is part BBa_K4212050. It is used to transform the B. subtilis knockoutstrain so as to produce a germinant receptor that contains a binding site forglucose, and when activated triggers an orthogonal signalling cascade resultingin germination.
References
[1]: Alzahrani, O. m., and A. Moir. “Spore Germination and GerminantReceptor Genes in Wild Strains of Bacillus Subtilis.” Journal of AppliedMicrobiology, vol. 117, no. 3, 2014, pp. 741–49. Wiley Online Library, https://doi.org/10.1111/jam.12566.
[2]: Iwanicki, Adam, et al. “A System of Vectors for Bacillus Subtilis Spore Surface Display.” Microbial Cell Factories, vol. 13, no. 1, Feb. 2014, p.30. BioMed Central, https://doi.org/10.1186/1475-2859-13-30.