Team:Shanghai HS ID/Description
Project Description
Lactobacillus Casei
Lactobacillus caseiis a lactic acid bacterium that could be found in various
natural or man-made environments [1]. It is used to make numerous day-to-day products including
cheese,
yogurt, and other dairy beverages. A study conducted in 2010 show that probiotics like L.Caseido contain
health promoting properties. Thirty-four adults were divided into two groups – one with a four-week intake
of probiotic fermented beverage, and one without. The results suggest that "probiotic fermented milk
beverage has an intestine-conditioning effect by improving the frequency of defecation and stool quality and
increasing the intrinsic bifidobacteria in healthy individuals with soft stool" [7]. As a probiotic,
L.caseiis also much safer and beneficial to the human body as a carrier for genetic materials than the
widely used E.colibacteria. [3]Making L.Caseihighly applicable in many frontiers of medicine,
pharmaceuticals, and food. However due to their restriction-modification system the bacteria’s
transformation rate is very inefficient, hence eminently restraining their capabilities.
Obstacles
L.Caseihas a self-protection mechanism called the restriction-modification system.
This system protects the bacteria through using restriction endonucleases to disable the invasive external
DNAs. However this leads to a low transformation efficiency when trying to introduce external DNAs in order
to maximizeL.Casei'scapabilities [2] This inefficiency hinders the study of other applications of
L.caseiin
fields such as probiotic-delivered vaccines[3]and other medicines. We discovered that traditional
methods
such as methylating foreign DNA or using high-temperatures to temporarily denature the restriction
modification system are not only time-consuming and costly but also not solving the problem fundamentally
[4][3]. Inspired by genetic engineering tools, our team came up with a new way of utilizing
CRISPR-Cas9 to
thoroughly solve this problem.
Solution
We constructed a plasmid, named pNCas9-LSEI-2094 equipped with a CRISPR-Cas9
complex in order to kick out the DNA segment, LSEI-2094 gene. This gene is involved in the synthesis of an
enzyme that is essential in the restriction-modification system [5]. After this modification, the
restriction enzyme could be temporarily inactivated so that the transferred exogenous DNA could successfully
avoid the restriction effect of the host bacteria (L. casei) restriction system. In other words, foreign
DNAs could be electrotransformed into L. casei ATCC 334 with higher efficiency and it is also expected to
express themselves easier in L. casei. With this modifiable L. casei ready to be tailored to our needs, the
benefits are immeasurable. The benefits of L. casei could come to fruition if the technology is applied to
popular and impactful industries such as pharmaceuticals and food.
Reference
1. Buriti, F. C., & Saad, S. M. (2007). Bactérias do grupo Lactobacillus casei:
caracterização, viabilidade como probióticos em alimentos e sua importância para a saúde humana [Bacteria of
Lactobacillus casei group: characterization, viability as probiotic in food products and their importance
for human health]. Archivos latinoamericanos de nutricion, 57(4), 373–380.
2. Matsumoto, K., Takada, T., Shimizu, K., Moriyama, K., Kawakami, K., Hirano, K.,
Kajimoto, O., & Nomoto, K. (2010). Effects of a probiotic fermented milk beverage containing Lactobacillus
casei strain Shirota on defecation frequency, intestinal microbiota, and the intestinal environment of
healthy individuals with soft stools. Journal of bioscience and bioengineering, 110(5), 547–552.
https://doi.org/10.1016/j.jbiosc.2010.05.016
3. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD):
National Library of Medicine (US), National Center for Biotechnology Information;1988. Available from:
https://www.ncbi.nlm.nih.gov/
4. Walsh, C. J., Healy, S., O'Toole, P. W., Murphy, E. F., & Cotter, P. D. (2020).
The probiotic L. casei LC-XCAL™ improves metabolic health in a diet-induced obesity mouse model without
altering the microbiome. Gut microbes, 12(1), 1704141. https://doi.org/10.1080/19490976.2020.1747330
5. 汪川 & 张朝武.(2008).以益生菌为载体的基因工程疫苗研究进展. 卫生研究(01),118-122.
doi:CNKI:SUN:WSYJ.0.2008-01-045.
6. 潘荣荣. 乳酸杆菌破壁方法及限制——修饰系统对转化效率影响的研究[D]. 扬州大学.
https://kns.cnki.net/KCMS/detail/detail.aspx?dbname=CMFD201801
7. Desirazu, N, Rao, et al. Type III restriction-modification enzymes: a historical
perspective[J]. Nucleic Acids Research, 2013, 42(1):45. https://doi.org/10.1093/nar/gkt616