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+ | <div class="sub-content"> | ||
+ | <div class="sub-title">Project Description</div> | ||
+ | <div class="article-title">Lactobacillus Casei</div> | ||
+ | <div class="article-content">Lactobacillus caseiis a lactic acid bacterium that could be found in various | ||
+ | natural or man-made environments <b>[1]</b>. 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" <b>[7]</b>. 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. <b>[3]</b>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.</div> | ||
+ | <div class="article-title">Obstacles</div> | ||
+ | <div class="article-content">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 <b>[2]</b> This inefficiency hinders the study of other applications of | ||
+ | L.caseiin | ||
+ | fields such as probiotic-delivered vaccines<b>[3]</b>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 | ||
+ | <b>[4]</b><b>[3]</b>. Inspired by genetic engineering tools, our team came up with a new way of utilizing | ||
+ | CRISPR-Cas9 to | ||
+ | thoroughly solve this problem. | ||
+ | </div> | ||
+ | <div class="article-title">Solution</div> | ||
+ | <div class="img-wrap no-margin"> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/2/26/T--Shanghai_HS_ID--chg1.jpg" alt="" /> | ||
+ | </div> | ||
+ | <div class="article-content">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. </div> | ||
+ | <div class="article-title"> | ||
+ | Reference | ||
+ | </div> | ||
+ | <div class="article-content">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.</div> | ||
+ | <div class="article-content">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</div> | ||
+ | <div class="article-content">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/</div> | ||
+ | <div class="article-content">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</div> | ||
+ | <div class="article-content">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.</div> | ||
+ | <div class="article-content">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</div> | ||
+ | <div class="article-content">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</div> | ||
+ | </div> | ||
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+ | <div class="footer-contact">Follow us on WeChat!</div> | ||
+ | <img class="footer-qrcode" src="https://static.igem.org/mediawiki/2021/c/c3/T--Shanghai_HS_ID--qrcode.png" /> | ||
+ | <p class="margin-bottom-10"><i>WeChat Official Account: 乳酸LAB</i></p> | ||
+ | <p>Contact Email:<i>jhxchen@gmail.com</i></p> | ||
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Latest revision as of 18:07, 19 October 2021
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
![](https://static.igem.org/mediawiki/2021/2/26/T--Shanghai_HS_ID--chg1.jpg)
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