Part:BBa_K3664024
cotG-GGGGS
This part is a sequence designed by us for the surface display of Bacillus subtilis. We provide the cotg gene sequence of the anchor protein CotG and an unpliable linker peptide.In use, we should construct such a structure as promoter sequence, our part, and target gene (removing termination codon). We can also add a fluorescent protein EGFP to help us verify.In our experimental design, we constructed the plasmid pdg1730 pcotg cotg yifh EGFP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
After the idea of using surface display technology, we learned some research progress of surface display technology through some review articles. We learned that foreign protein and peptide targeting and anchoring on phage and extracellular surface are becoming more and more important, and have been used as basic and applied research tools in microbiology, molecular biology, vaccinology and Biotechnology. The spores of Bacillus subtilis are surrounded by a spore shell. Some proteins have been identified in the outer layer and have been successfully used to immobilize antigens and other proteins and enzymes.[1]
Through reading a large number of literature, we believe that surface display technology can be applied to our project, and Bacillus subtilis surface display technology [2] is the most feasible and operational.
There is a wide variety of ways to display a protein outside spores of B.subtilis. After studying several reviews, we found the following strategies for spore display:
We designed three main structures: anchor protein, linker peptide and target gene. At the same time, we combined a fluorescent protein GFP to verify the work.In the work of searching for suitable anchoring proteins, we found that cotG are widely used in catalysis.
Given that recombinant method in only applied in the field of vacancies research, we opt for the integrate method. However, it is hard to determine whether to express over protein from episomal vectors or from genome. Nevertheless, both method necessitate the selection of vectors. Hence, we started to select vectors regardless of their ability for crossovers. Episomal vectors have more replicas, which provides a higher expression level, whilst genome expression is more stable, having no risk of losing target genes. Based on our knowledge, pJS700（[4][5][6]） (used for episomal expression) and pDG1730([7][8][9]) (used for genome expression) are the most widely used and reasonable vectors regarding to display of enzyme. Considering that pJS700 is harder to purchase, we decided to chose pDG1730. This also means that we are going to use a double crossover system, which integrates our designed gene to B.subtilis’ genome through a double crossover reaction:
Since our scope has narrowed down to a specific construction system---double crossover recombinant strategy, we started searching for the specific route for construction the spore display system. It is widely accepted that the key for molecular biology as well as enzyme industry is to determine genotypes and protein sequence. Multiple review shows that the basic structure for the expression unit is as follow:
Promoter of anchor protein-CDS of anchor protein-link-CDS of gene
We also reached for exact cases of spore display, which verifies the above structure:
common structure(structure of vector compiled from several article)([3][10])：
exchange arm 1 ——N '- CotG / C promoter, RBS, structural gene (without stop codon) - C' ——n '- target gene (with stop codon) - C' ——screening marker —— exchange arm 2
But there are differences in different structural designs.
（1）Whether there is a spacer between the anchor protein and the target gene.
（2）Whether there is a linker.
（3）The direction of integration into the carrier is different.
（4）The number of expression cassettes in series was different.
（5）The number of CDs in tandem was different.
Consequently, the key point in this stage is to chose an proper anchor protein and peptide link.
All the possible anchor protein and peptide link along with their application is reported in several review:
Generally speaking cotG is most widely used when displaying enzymes. Thus, it is obvious that the cotG is the best candidate. Some research also demonstrates that cotC, especially co-expression of cotG and cotC result in a high yield of enzyme reaction. However, we only testified the former given that our time is limited. A flexible link (usually with one relicus) GGGGS provides a higher enzyme activity compared with a stiff link or no link at all. However, given that our former research reveals that stiff link (EAAAK) is instrumental when expressing recombinant enzyme. Hence we decided to try both links.
Now we will take one of the most feasible designs as one of our parts.
In the absence of experimental verification, combined with the literature data,an unpliable link peptide (GGGGS) is commonly used, we believe that its feasibility is the highest. At the same time, we also add a EGFP fluorescent protein gene, hoping that in future experiments, we can detect the fluorescence intensity to help us verify whether it is successful. pDG1730-pcotG-cotG-yifH-GFP
Protein sequence:
Tips:
(1)The orange bold part is yifH sequence(genebank:QHQ38277.1);
(2)The blue part is CotG sequence(genebank:YP_009514005.1);
(3)The green part is EGFP sequence;
(4)The red part shows the link peptide;
Nucleic acid sequence:
Tips：
(1)The bold black sequences are cotg sequences without codon optimization(genebank:YP_009514005.1);
(2)The bold yellow sequence is the yifH sequence after codon optimization(genebank:QHQ38277.1)（Optimization for B.subtilis);
(3)The green part is the EGFP sequence after codon optimization for B. subtilis（genebank: YP_009062989.1）;
(4)The red letter indicates the link peptide;
(5)The underlined part is the restriction site, BamHI / HindIII (with protective base);
(6)The blue word sequence is CotG promoter;
(7)The start codon and stop codon are in bold purple.
Plasmid map:
[1]Junehyung Kim,Wolfgang Schumann.Display of proteins on Bacillus subtilis endospores.Cell. Mol. Life Sci. (2009) 66:3127–3136
[2]Ping Lin, Haibo Yuan.Progress in research and application development of surface display technology using Bacillus subtilis spores.Applied Microbiology and Biotechnology
[3]Guoyan Zhang.Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications.J. Microbiol. Biotechnol. (2019), 29(2), 179–190
[4]Guohui Li.Display of Bombyx mori Nucleopolyhedrovirus GP64 on the Bacillus subtilis Spore Coat.Curr Microbiol (2011) 62:1368–1373
[5]Langyong Mao.Surface Display of Human Serum Albumin on Bacillus subtilis Spores for Oral Administration.Curr Microbiol (2012) 64:545–551
[6]Nan Wang.Display of Bombyx mori Alcohol Dehydrogenases on the Bacillus subtilis Spore Surface to Enhance Enzymatic Activity under Adverse Conditions.PLoS ONE 6(6): e21454. doi:10.1371/journal.pone.0021454
[7]Youvraj Sohni.Cloning and development of synthetic internal amplification control for
Bacillus anthracis real-time polymerase chain reaction assays.Diagnostic Microbiology and Infectious Disease 61 (2008) 471–475
[8]Anne-Marie Gu6rout-Fleury.Plasmids for ectopic integration in Bacillus subtilis .Gene 180 (1996) 57-61
[9]Hongling Liu.Production of trehalose with trehalose synthase expressed and displayed on the surface of Bacillus subtilis spores.Liu et al. Microb Cell Fact (2019) 18:100
[10]Le H. Duc.Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen.V accine 25 (2007) 346–355
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