Part:BBa_K4212039
ChiS6
L1
Our Experiment Design
Amplification Level 0 Parts
Due to issues of both length and synthesis complexity, the coding sequence for our chitinase of choice (ChiS) was ordered from IDT as two separate gblock segments, alongside anchor protein CotZ (ordered instead as a single fragment).
The anchor protein CotG was instead PCR amplified from the genome in two versions, one featuring the custom-designed linker and one without. Indeed, the same forward primer was used in both amplifications, but in one case the reverse one featured the linker sequence in addition to the standard overhang.
The two chitinase fragments and the primers used for the amplification of CotG were specifically designed to feature recognition sites of restriction enzyme BpiI, the toolkit’s enzyme of choice for L0 assembly, and appropriate overhangs to allow for correct directional assembly. This allowed the successful assembly of the anchor protein, linker and chitinase into a single fusion protein, contained in the toolkit’s L0 construct reserved for coding sequences (0c).
Amplification Level 1 Parts
The newly obtained L0 CDS were subsequently assembled together with a promoter, RBS and terminator into a L1 construct (1A), containing an ampicillin resistance in E. coli, to yield a functional transcriptional unit. However, the L1A backbones in the toolkit do not feature neither an origin of replication functional in B. subtilis nor an antibiotic marker. Thus, it was necessary to use an EXP L1 vector, so dubbed in the toolkit, which did contain these features. However, one challenge present was the presence of the same antibiotic marker in the L0 vector and EXP L1, which made screening of correctly assembled L1 plasmids quite challenging.
Accounting for this feature of the toolkit we performed three different strategies in parallel:
1) Assembly with extensive screening rounds of L0 parts into STK108 (EXP L1)
2) Assembly of L0 parts into L1A backbone (change of antibiotic marker from chloramphenicol to ampicillin) and then from L1A to STK110 (EXP L2), featuring a change from Amp to Cm.
3) Assembly in pMAD L2 vector for genomic integration – must be L2 to include homology arms, must also include an antibiotic marker TU to differentiate from L1s (both have Amp resistance)
In all cases, the composition of our transcriptional unit stayed the same, namely with: - Constitutive promoter: (Hyperspank) - RBS: tmRBS1 (stk45) - CDS: L0 Fusion Protein CDS (CotG-ChiS/CotG-L-ChiS/CotZ-ChiS) - Terminator: L3S2p21 (STK077)
We chose to construct the fusion protein with and without the linker. According to the modeling prediction, the flexible linker can help to reduce steric hindrance between each component so that the proteins can be better fused together. However, we would like to verify the predictions by experiments and then we will select the better one for chitinase display.
Our Experiment Results
Given the tightly controlled process of sporulation, we also wanted to test the difference in burden, expression level and functionality using an anchor protein specific promoter vs. a constitutive one. One concern was that using a constitutive promoter might result in excessive metabolic load, affecting the standard functioning of the cell including sporulation. Furthermore due to the timeliness of CotG expression, another worry was that the fusion protein might integrate into the wrong layer of the spore coat. Thus, we designed a new part for the STK toolkit, a CotG native promoter + rbs. We accomplished this by taking 200bp upstream from the CotG CDS in the B. subtilis genome and adding the appropriate recognition sites and overhangs on either end.
As can be seen by the figure below, we obtained a positive band for CotG-ChiS, CotG-L-ChiS ,and CotZ-ChiS. We then inoculated these clones, miniprepped them and sent them for Sanger sequencing. Clones that aligned perfectly to the in-silico design (no mutations), were then used in subsequent assembly levels and stored in glycerol stocks.
Usage and Biology
It was shown by previous literature that the CotG coat protein of B.Subtilis can be used to display other proteins, which provide additional functions. Chitinase is an enzyme, which can break down chitin into N-acetyl-glucosamine (NAG): being able to break down chitin polymers into individual monomers and being able to sense them to indicate the presence of a fungal pathogen is a crucial preliminary aspect of our fungicidal response.
References
[1] Elieh-Ali-KomiD, Hamblin MR. Chitin and Chitosan: Production and Application of VersatileBiomedical Nanomaterials. Int J Adv Res (Indore). 2016 Mar;4(3):411-427. Epub2016 Mar 1. PMID: 27819009; PMCID: PMC5094803.
[2] Anand Nagpure, Bharti Choudhary &Rajinder K. Gupta (2014) Chitinases: in agriculture and humanhealthcare, Critical Reviews inBiotechnology, 34:3, 215-232, DOI: 10.3109/07388551.2013.790874
[3] Hamid R, Khan MA, Ahmad M, Ahmad MM,Abdin MZ, Musarrat J, Javed S. Chitinases: An update. J Pharm Bioallied Sci.2013 Jan;5(1):21-9. doi: 10.4103/0975-7406.106559. PMID: 23559820; PMCID:PMC3612335.
[4] ina Adrangi, Mohammad Ali Faramarzi,From bacteria to human: Ajourney into the world of chitinases, Biotechnology Advances, Volume 31, Issue8, 2013, Pages 1786-1795, ISSN 0734-9750, https://doi.org/10.1016/j.biotechadv.2013.09.012.
[5]McKenney, P., Driks, A. & Eichenberger, P. The Bacillus subtilis endospore:assembly and functions of the multilayered coat. Nat Rev Microbiol 11,33–44 (2013). https://doi.org/10.1038/nrmicro2921
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1062
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1062
Illegal NheI site found at 206 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1062
Illegal BglII site found at 101
Illegal BamHI site found at 1047 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1062
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1062
- 1000COMPATIBLE WITH RFC[1000]
| None |