Difference between revisions of "Team:Nagahama/Introduction"

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Introduction
 
Introduction
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１． Last year, we devised Flavorator which preserve food by fragrance component with antibacterial effect and introduced the concept of it.
  
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Last year we introduced the concept of Flavorator.
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２．We chose geraniol and farnesol as the fragrance that there was an antibacterial effect . Then We succeed in letting at E.coli compose them.
  
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Here, we explain Flavorator's concept.  
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3.But synthetic amount of geraniol and farnesol was very low we cannot put Flavorator to practical use
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4.So, we tried to select only and increase farnesol as fragrance with antibacterial action E.coli creates to improve practicality of Flavorator in this year.
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5,There are three reason that focus on farnesol.
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(6）1,Farnesol does not impair  flavor of  food because it is almost odorless.
  
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Food problems are serious matters in the world.
 
  
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Among them is food preservation.
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7  2.Farnesol has a high antifungal activity
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8,Third reason is Escherichia coli can synthesize of farnesol in a process that it has from original.
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9.As a method for Farnesol production increase, we tried to increase of farnesol synthesis amount by adding more genes in farnesol synthesis device of the last year.
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10. We introduce three gene to “E.coli”.
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11. 1.Gene that increases the intermediate product of farnesol synthesis
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12.1-deoxy-D-xylulose 5-phosphate (DXP) which is used for farnesol synthesis is also used for other compound synthesis.
  
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Ideal food preservation is keeping food without causing quality change for a long time and at low cost.  
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13,Therefor we added a new DXP synthase to increase the intermediate product.
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14.2, dephosphorylation enzyme gene
  
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Various preservation methods have been used so far, and we created a new one.
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（15）Synthesis of farnesol depends on farnesol precursor  to endogenous dephosphorylation enzyme of E. coli, last year.
  
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It is "Flavorator"!!
 
  
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Thirty years ago, “KOZOKO（香蔵庫 in Japanese）” was proposed by Professor Yozo Iwanami.  
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16.It has introduced an additional endogenous phosphatase gene (YbjG, PgpB) in E. coli in order to increase the production of farnesol this year.
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（17）3. A gene for improve resistance to farnesol of E. coli.
  
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The name “KOZOKO” can be directly translated into ‘flavor (=KO, 香)’- ‘preserved (=ZO, 蔵)’- ‘box (=KO, box)’.
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18,Antibacterial effect of farnesol kill E.coli. And, E. coli will die by farnesol made with myself.
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However, “KOZOKO” is not just a flavor-keeping box but a box to preserve food in a fragrance.  
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19.So, we made Escherichia coli overexpress gene of endogenous (marA), an activator gene of AcrAB-TolC efflux pump．
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Therefore it improves the resistance to farnesol.
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(（20）We thought about the method to improve practicality of Fravorator without using recombinant .
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（21.If you are trying to fill the farnesol in Flavorator, there is a possibility allowed to escape the recombinant E. coli to the outside.
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22.To improve practicality of Flavorator, it is necessary for E.coli to be recombinant.
  
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Then, what flavors should we put in KOZOKO?
 
  
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The flavors should have antimicrobial or insecticidal activities.  
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23.We attempted to increase production of farnesol using CRISPER / Cas9.
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The best candidates are those of plant origin.
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Plants cannot move to other places even when microbes or insects attack them.
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So, they produce antimicrobial or insecticidal agents.
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These agents are mostly not harmful to humans and animals.
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Therefore, “KOZOKO” with antimicrobial flavors would be an energy-saving substitute for an ordinary electric refrigerator.
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In order to make “KOZOKO” practical two problems have to be solved.
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First, we must select appropriate flavors and plants that can be cultivated under the various climate conditions.
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Second, cost issues must be cleared.
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Mass cultivation of plants and extraction of pure flavors from plants are time-consuming and expensive.
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Consequently, at present “KOZOKO” is under the state of conceptual idea.
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We created a new version of “KOZOKO” utilizing syntheticbiology.
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We named it “Flavorator”.
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And we want to solve Food problem all over the world!
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Recombinant ''Escherichia coli''produces antimicrobial and insecticidal volatile gaseous substances that suppress bacterial growth in the “Flavorator” and prevent insects from entering the “Flavorator”.
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In the future,　“Flavorator” can slow down food decay and solve one of the food problems.
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This year, we started an action towards improvement of Flavorator.
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We use ''E. coli''as a model organism to show feasibility of Flavorator.
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We decided to do the following.
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First, we use high antimicrobial fragrance to synthesis easily in ''E. coli''.
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Second, we need to optimize ''E.coli'' to Flavorator.
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○First, we chose farnesol as an effective fragrance which can be synthesized easily by ''E.coli''.
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.
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There are three reasons for this choice.
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1. We can increase production of farnesol by overexpression of ''E.coli''s endogenous gene because ''E. coli'' can synthesize farnesol in theory.
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24.E. coli has a pathway that originally make the farnesol.
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We tried to knockout gdhA, gene did not relate to falnesol synthesis, with CRISPER-CAS9 and be accumulate intermediate metabolite.
 
[[File:経路図.png|800px|thumb|left|Fig1: Metabolic pathway of ''Escherichia coli''.]]
 
[[File:経路図.png|800px|thumb|left|Fig1: Metabolic pathway of ''Escherichia coli''.]]
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2. Farnesol does not impair flavor of food because it is almost odorless.
 
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3. Farnesol has a high antibacterial activity against food poisoning bacteria.
 
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◯Second, we did three designs ,upon optimization of ''E.coli''.
 
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1. Construction of farnesol synthetic plasmid for ''E. coli''
 
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[[File:1プラスミド.png|800px|thumb|left|Plasmid Construction]]
 
[[File:1プラスミド.png|800px|thumb|left|Plasmid Construction]]
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Endogenous phosphatase of Escherichia coli is not able to synthesize much farnesol.
 
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Therefore, we introduced additional endogenous farnesol synthesis enzyme genes which are PgpB and YbjG in ''E.coli''.
 
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We added PgpB and YbjG to device of the last year
 
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Ybjg and PgpB encode farnesol synthase.
 
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Farnesol synthase dephosphorylates farnesyl diphosphate.
 
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As a result, this device can synthesize farnesol.
 
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2. Improved resistance of ''E. coli'' to farnesol
 
  
 
[[File:プラスミド2.png|800px|thumb|left|alt text]]
 
[[File:プラスミド2.png|800px|thumb|left|alt text]]
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We further introduce an activator gene of AcrAB-TolC efflux pump (MarA) to We further introduce an activator gene of AcrAB-TolC efflux pump (MarA).
 
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This improves the resistance of ''E. coli'' against farnesol.
 
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3. Addition of metabolic pathway from xylose genes.
 
  
 
[[File:経路図2.png|800px|thumb|left|alt text]]
 
[[File:経路図2.png|800px|thumb|left|alt text]]
  
 
[[File:プラスミド3.png|800px|thumb|left|alt text]]
 
[[File:プラスミド3.png|800px|thumb|left|alt text]]
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The ''m-ribB'' (1-Deoxy-D-xylulose 5-phosphate synthase gene) which is mutant of ''ribB'' mutated G108S 1-Deoxy-D-xylulose 5-phosphate synthase can utilize D-ribulose 5-phosphate as substrates, generating 1-Deoxy-D-xylulose 5-phosphate.
 
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''Ybjg'' and ''PgpB'' encode farnesol synthase.
 
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Farnesol synthase dephosphorylates farnesyl diphosphate.
 
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As a result, this device can synthesize farnesol.
 
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The three designs above are methods to transform plasmids into ''E. coli''.
 
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In other words, we use these as recombinants to "Flavorator".
 
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We are forbidden to put ''E.coli'' outside of the laboratory.
 
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It is difficult to realize "Flavorator" the way things are going.
 
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So we used the CRISPR-Cas9 to alter the genome of ''E. coli''.
 
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Accordingly, we thought preventing recombinant leaking to the outside of the laboratory.
 
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For example, we thought about trying to knock out the genes involved in the growth of ''E. coli'', so that ''E. coli'' cannot grow except for a particular medium.
 
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We tried to realize "Flavorator" in the above-described method.
 
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Therefore, we do knockout of an endogenous gene of ''E. coli'' using the CRISPR-Cas9.
 
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By doing this we thought about trying to make more suitable ''E. coli'' strain to "Flavorator".
 
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Specifically, we had knocked out unwanted intermediate metabolite synthase gene to synthesize fragrance without incorporating the plasmid.
 
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So we tried to knockout one gene (gdhA) to explain this concept.
 
  
 
[[File:経路図3.png|800px|thumb|left|alt text]]
 
[[File:経路図3.png|800px|thumb|left|alt text]]
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''Escherichia coli'' originally synthesizes little farnesol.
 
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Therefore, we knocked out intermediate metabolite synthase gene unnecessary for farnesol synthesis in order to facilitate the synthesis proceeding to DXP which is the substrate of farnesol.
 
  
 
[[File:プラスミド4.png|800px|thumb|left|alt text]]
 
[[File:プラスミド4.png|800px|thumb|left|alt text]]
  
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CRISPR system is a bacterial immune system which is used as a gene engineer machine now.
 
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It has three types and we chose the type II CRISPR system.
 
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This system contains three parts.
 
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First, this system can express two kinds of single strand RNA: tracrRNA and crRNA.
 
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crRNA’s structure is like “repeat---spacer---repeat”.
 
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The spacer part is complementary to target gene’s DNA sequence, and the repeat is complementary to tracrRNA.
 
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tracrRNA can also have interaction with a protein coded in this system --- Cas9, an endonuclease.
 
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Once we modified the spacer sequence of a CRISPR system and transformed it into bacteria, it will search for target gene Intermediate metabolite synthase gene was not used to synthesize farnesol gene in our project) in bacteria’s genome by crRNA.
 
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Once the target is found, Cas9 protein will bind on this gene’s DNA sequence with the help of tarcrRNA and crRNA.
 
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Then, Cas9 will make a small double strand break on the gene.
 
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So this gene’s sequence will be modified by later homozygous recombination, resulting in the knock-out of this gene.
 
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We have considered trying to be able to use as a strain for Flavorator by changing the genome of ''E. coli'' strain.
 
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We showed the possibility to synthesize fragrance without recombinant by using CRISPR-Cas9.
 
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In the future, we will not need to use recombinant ''E. coli''.
 
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Revision as of 01:57, 20 October 2016

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Introduction １． Last year, we devised Flavorator which preserve food by fragrance component with antibacterial effect and introduced the concept of it.

２．We chose geraniol and farnesol as the fragrance that there was an antibacterial effect . Then We succeed in letting at E.coli compose them.

3.But synthetic amount of geraniol and farnesol was very low we cannot put Flavorator to practical use 4.So, we tried to select only and increase farnesol as fragrance with antibacterial action E.coli creates to improve practicality of Flavorator in this year. 5,There are three reason that focus on farnesol. (6）1,Farnesol does not impair flavor of food because it is almost odorless.


7 2.Farnesol has a high antifungal activity 8,Third reason is Escherichia coli can synthesize of farnesol in a process that it has from original. 9.As a method for Farnesol production increase, we tried to increase of farnesol synthesis amount by adding more genes in farnesol synthesis device of the last year. 10. We introduce three gene to “E.coli”. 11. 1.Gene that increases the intermediate product of farnesol synthesis 12.1-deoxy-D-xylulose 5-phosphate (DXP) which is used for farnesol synthesis is also used for other compound synthesis.

13,Therefor we added a new DXP synthase to increase the intermediate product. 14.2, dephosphorylation enzyme gene

（15）Synthesis of farnesol depends on farnesol precursor to endogenous dephosphorylation enzyme of E. coli, last year.


16.It has introduced an additional endogenous phosphatase gene (YbjG, PgpB) in E. coli in order to increase the production of farnesol this year. （17）3. A gene for improve resistance to farnesol of E. coli.

18,Antibacterial effect of farnesol kill E.coli. And, E. coli will die by farnesol made with myself. 19.So, we made Escherichia coli overexpress gene of endogenous (marA), an activator gene of AcrAB-TolC efflux pump． Therefore it improves the resistance to farnesol. (（20）We thought about the method to improve practicality of Fravorator without using recombinant . （21.If you are trying to fill the farnesol in Flavorator, there is a possibility allowed to escape the recombinant E. coli to the outside. 22.To improve practicality of Flavorator, it is necessary for E.coli to be recombinant.


23.We attempted to increase production of farnesol using CRISPER / Cas9.

24.E. coli has a pathway that originally make the farnesol. We tried to knockout gdhA, gene did not relate to falnesol synthesis, with CRISPER-CAS9 and be accumulate intermediate metabolite.

Fig1: Metabolic pathway of Escherichia coli.
Plasmid Construction
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REFERENCES

[http://onlinelibrary.wiley.com/doi/10.1002/biot.201600250/abstract?systemMessage=Wiley+Online+Library+will+be+unavailable+on+Saturday+30th+July+2016+from+08:00-11:00+BST+/+03:00-06:00+EST+/+15:00-18:00+SGT+for+essential+maintenance.Apologies+for+the+inconvenience [1] Wang C, Park JE, Choi ES, Kim SW.(2016).Farnesol production in Escherichia coli through the construction of a farnesol biosynthesis pathway – application of PgpB and YbjG phosphatases. Biotechnology Journal 11(10): 1291–1297.

[http://aem.asm.org/content/81/1/130.short [2] Kirby J, Nishimoto M, Chow W. N.R, Baidoo E.E., Wang G, Martin J, … & Keasling J D.(2015).Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate.Applied and Environmental Microbiology,81(1), 130-138.

[http://link.springer.com/article/10.1007/s00284-009-9408-9 [3]Gomes FIA, Teixeira P, Azeredo J, Oliveira R. (2009). Effect of Farnesol on Planktonic and Biofilm Cells of Staphylococcus epidermidis. Current Microbiology, 59(2), 118-122.

[http://www.nature.com/nbt/journal/v31/n3/full/nbt.2508.html%3FWT.ec_id%3DNBT-201303 [4]Jian W, Bikard D, Cox D, Zhang F & Marraffini LA. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233-239.

[http://www.sciencedirect.com/science/article/pii/S1096717615000750 [5]Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang Y, Chen T, Zhao X.(2015). Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing. Metabolic Engineering, 31, 13-21.

[http://www.sciencedirect.com/science/article/pii/S1389172312004185 [6]Shah A A, Wang C, Chung Y R, Kim J Y, Choi E S, Kim S W. (2013).Enhancement of geraniol resistance of Escherichia coli by MarA overexpression. Journal of Bioscience and Bioengineering, 115(3),235–258.

[http://nar.oxfordjournals.org/content/early/2016/04/08/nar.gkw223.abstract [7]Cui L, Bikard D. (2016).Consequences of Cas9 cleavage in the chromosome of Escherichia coli. Nucleic acids research, gkw223.

[http://www.sciencedirect.com/science/article/pii/S1096717605000741 [8]Yuan L.Z, Rouvière P.E, LaRossa R.A, Suh W. (2006).Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metabolic Engineering 8(1) 79–90.

[http://aem.asm.org/content/81/15/5103.short [9]Pyne ME, Moo-Young M, Chung DA, Chou CP. 2015. Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli. Applied and Environmental Microbiology 81:5103–5114.