Difference between revisions of "Team:Aachen/Description"

(→‎How We Improved a Previously Existing BioBrick)
 
(26 intermediate revisions by 5 users not shown)
Line 1: Line 1:
 
{{Team:Aachen/Header}}
 
{{Team:Aachen/Header}}
 +
 +
{{Team:Aachen/StartTour}}
  
 
These days, the whole bioeconomy is dependent on one valuable resource: sugars. No matter if they are produced from starch or cellulose, they always originate from plants. But while the global demand for carbon sources is growing, the arable land is shrinking and droughts are more frequent.
 
These days, the whole bioeconomy is dependent on one valuable resource: sugars. No matter if they are produced from starch or cellulose, they always originate from plants. But while the global demand for carbon sources is growing, the arable land is shrinking and droughts are more frequent.
 +
  
 
At iGEM Aachen, we aim to make the bioeconomy independent from plant-derived sugars. We do this by teaching ''E. coli'' to use '''methanol''' as a carbon source and convert it to '''glycogen''', the bacterial equivalent to starch.  
 
At iGEM Aachen, we aim to make the bioeconomy independent from plant-derived sugars. We do this by teaching ''E. coli'' to use '''methanol''' as a carbon source and convert it to '''glycogen''', the bacterial equivalent to starch.  
  
 
Therefore, we introduce a '''synthetic pathway''', implementing it ''in vivo'' for the first time.
 
Therefore, we introduce a '''synthetic pathway''', implementing it ''in vivo'' for the first time.
 +
  
 
For this kind of metabolic engineering research, chemostat cultivation of candidate strains is essential. However, traditional continous cultivations are very costly and not affordable for everyone. To solve this problem, we are developing a '''do-it-yourself bioreactor''' with very low culture volume. This bioreactor is accompanied by '''hardware''' and '''software''' that is cost-effective and user-friendly.
 
For this kind of metabolic engineering research, chemostat cultivation of candidate strains is essential. However, traditional continous cultivations are very costly and not affordable for everyone. To solve this problem, we are developing a '''do-it-yourself bioreactor''' with very low culture volume. This bioreactor is accompanied by '''hardware''' and '''software''' that is cost-effective and user-friendly.
−
 
−
'''something about practices is missed here.'''
 
−
 
−
 
−
 
−
 
−
''inspiration from Design Page:''
 
−
 
−
=Introduction=
 
−
 
−
Products from the bioeconomy play an important role in a sustainable future, but are dependent on cheap and available biomass as a carbon source. The availbality of sustainable biomass though, is limited by the arable area of our planet. Even by deforestation and setting up new agricultural area, we cannot provide enough arable land for both biomass for the bioindustry and food crops to solve the problem of world hunger. So if we hardly manage to feed the population, how can we guarantee the supply of biomass for other biotechnological products?
 
−
 
−
 
−
Another important aspect to consider is that most of our everyday products are mainly based on fossil resources. The main source for sustainable products today, are plants. However, they are really inefficent in fixing CO2 and need a lot of time, water and space.
 
  
  
−
The best option would be to use CO2 from the air to form products. Recent technological advancements made it possible to convert CO2 into methanol with an incredible efficieny. This is where our project comes in. To take a step forward in solving this complex problem, we developed our ''E. coli'' that converts methanol to glycogen.  
+
Concering the DIY principle of our modular selfmade bioreactor we established contacts to community labs to discuss about the developement of the European '''Community Lab Scene'''.
  
 +
Regarding biosafety in the lab we tested and discussed our new '''documentation system''' with other teams to explore the technology and experiences.
  
−
Glycogen is a sugar polymer that is safe to handle and can be easily converted into glucose. Therefore it can then be used as a carbon source for almost all existing bioprocessess since those rely on sugar. Our project has the potential to make the bioindustry independent from plants and instead use CO2 from the air. Thereby not only methanol is opened up as a new carbon source but also the CO2 level in the atmosphere is reduced. We connect the bioeconomy with advanced technologies of CO2 fixation.  
+
<html><div class="col-md-12"></html>
 +
{{Team:Aachen/ReadMore|title=Safety|link=/Team:Aachen/Practices/Safety|picture=rmsafety|url=/wiki/images/0/04/Aachen_tile_HumanPractice_Overview_Safety2.JPG}}
  
 +
{{Team:Aachen/ReadMore|title=tubefront|link=/Team:Aachen/Practices/tubefront|picture=rmTubefront|url=/wiki/images/8/84/Aachen_tile_HumanPractice_Overview_Tubefront.jpg}}
  
−
We decided to use the newly developed, ATP-neutral Methanol Condensation Cycle to enable E.coli to take up methanol. Glycogen accumulation was achieved by knocking out the degradation enzymes and overexpressing the synthesis genes. Therefore we developed a functioanl ''glgCAB'' glycogen synthesis operon, which successfully produces glycogen and is our first functional prototype.
+
{{Team:Aachen/ReadMore|title=Human Practice|link=/Team:Aachen/Practices|picture=rmCollaborations|url=/wiki/images/b/b9/Aachen_CL10.jpg}}
  
 +
{{Team:Aachen/ReadMore|title=Bioreactor|link=/Team:Aachen/Lab/Bioreactor|picture=rmBioreactor|url=/wiki/images/1/1a/Aachen_tile_Lab_Overview_Bioreactor.JPG}}
  
−
Our product adresses the problem in a multifunctional way because not only can the potential of methanol be exploited but also the bioeconomy can get independent of plants and there will be no competition of resources for industry and for bioindustry.
+
{{Team:Aachen/ReadMore|title=Glycogen|link=/Team:Aachen/Lab/Glycogen|picture=rmGlycogen|url=/wiki/images/4/46/Aachen_tile_Lab_Overview_Glycogen.JPG}}
−
Other potential solutions...
+
  
 +
{{Team:Aachen/ReadMore|title=Methanol|link=/Team:Aachen/Lab/Methanol|picture=rmMethanol|url=/wiki/images/9/93/Aachen_tile_Lab_Overviwe_Methanol.JPG}}
  
  
 +
<html></div><span style="color:transparent;line-heigth:0em;">a</span></html>
 +
=Motivation=
  
 +
When we first started with extensive literature research at the beginning of the project, we came across an interesting paper about an ATP neutral cycle for methanol uptake. <ref>Bogorad IW, Chen CT, Theisen MK, Wu TY, Schlenz AR, Lam AT, Liao JC. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15928-33. doi: 10.1073/pnas.1413470111. Epub 2014 Oct 29. PubMed PMID: 25355907; PubMed Central PMCID: PMC4234558.</ref>  Right from the beginning we saw the great potential of methanol as a carbon source that is currently produced in large amounts but only partially used for fuels and not nearly up to its full potential. At this point we had the base for our project idea. It was further developed by research on other sources of methanol. We investigated the technical fixation of CO{{sub|2}} from the air and found out that methanol can easily be produced with this method as it is already by some companies.<ref>http://www.sunfire.de/en/</ref> But what comes next after methanol uptake and how can we make it available for various industries? The answer is sugars. In the bioeconomy most processes rely on sugar and therefore we decided to convert the methanol into glycogen, the storage molecule of sugar in bacteria. This contributes to making the bioindustry independent of plants and instead rely on renewable methanol.
  
  
 +
=How We Improved a Previously Existing BioBrick=
  
−
{{Team:Aachen/Footer|color=green}}
+
Enhancing the glycogen accumulation in ''Escherichia coli'' is an important aspect of our project. In this context we developed our '''glycogen synthesis operon''' (''glgCAB'').
−
<p>
+
It is a composite part that combines all 3 glycogen formation enzymes. The ADP-glucose pyrophophorylase (GlgC) forms ADP-glucose from ATP and glucose-1-phosphate, the glycogen synthase (GlgA) elongates α-1,4-linked chains and the branching enzyme (GlgB) catalyzes the formation of α-1,6-linked branches. The GlgC is based on the part [http://parts.igem.org/Part:BBa_K118016 BBa_K118016] from Team Edinburgh 2008, but the RBS B0034 was added to the existing Biobrick. Since we could prove that strains expressing our polycistronic construct accumulate more glycogen than strains just expressing ''glgC'', this part is an extension and improvement of the existing Part BBa_K118016.
−
Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
+
−
</p>
+
  
  
−
<br />
+
The construct was confirmed by sequencing. The expression of all three enzymes GlgC, GlgA and GlgB was tested in BL21 Gold (DE3) strains containing BBa_K1585321 in a pSB1A30 expression vector.
−
<h4>References</h4>
+
−
<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you.</p>
+
  
  
 +
{{Team:Aachen/Figure|Aachen_glgCAB for registry.png|title=SDS-PAGE of ''glgCAB'' in pSB1A30|subtitle=''glgCAB'' in pSB1A30 was expressed in BL21 Gold (DE3) strains and IPTG induced. The small arrows indicates the expected bands for all three enzymes . The BL21 Gold (DE3) wild type was used as the negative control.|size=large}}
  
−
<h4>Inspiration</h4>
+
The combined functionality was characterized by iodine staining (see picture below). It was performed with Lugol's iodine which dyes glycogen in a brownish color. If more glycogen is present, the color of stainend cultures is darker. The darker staining of BL21 Gold (DE3) transformants of BBa_K1585321 indicates considerably more glycogen accumulations compared to the wild type.
−
<p>See how other teams have described and presented their projects: </p>
+
  
−
<ul>
+
{{Team:Aachen/Figure|Aachen_glgCAB , WT_v2.png|title=Iodine staining BL21 Gold (DE3) + ''glgCAB'' vs. wild type |subtitle=Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) + ''glgCAB''  stained distinctly darker than the BL21 Gold (DE3) wild type.|size=medium}}
−
<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
+
−
<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
+
−
<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
+
−
</ul>
+
  
−
</div>
+
=References=
−
</html>
+
<references/>
 +
{{Team:Aachen/Footer|color=red}}

Latest revision as of 01:58, 19 September 2015

Start the Journey !

These days, the whole bioeconomy is dependent on one valuable resource: sugars. No matter if they are produced from starch or cellulose, they always originate from plants. But while the global demand for carbon sources is growing, the arable land is shrinking and droughts are more frequent.


At iGEM Aachen, we aim to make the bioeconomy independent from plant-derived sugars. We do this by teaching E. coli to use methanol as a carbon source and convert it to glycogen, the bacterial equivalent to starch.

Therefore, we introduce a synthetic pathway, implementing it in vivo for the first time.


For this kind of metabolic engineering research, chemostat cultivation of candidate strains is essential. However, traditional continous cultivations are very costly and not affordable for everyone. To solve this problem, we are developing a do-it-yourself bioreactor with very low culture volume. This bioreactor is accompanied by hardware and software that is cost-effective and user-friendly.


Concering the DIY principle of our modular selfmade bioreactor we established contacts to community labs to discuss about the developement of the European Community Lab Scene.

Regarding biosafety in the lab we tested and discussed our new documentation system with other teams to explore the technology and experiences.


a

Motivation

When we first started with extensive literature research at the beginning of the project, we came across an interesting paper about an ATP neutral cycle for methanol uptake. [1] Right from the beginning we saw the great potential of methanol as a carbon source that is currently produced in large amounts but only partially used for fuels and not nearly up to its full potential. At this point we had the base for our project idea. It was further developed by research on other sources of methanol. We investigated the technical fixation of CO2 from the air and found out that methanol can easily be produced with this method as it is already by some companies.[2] But what comes next after methanol uptake and how can we make it available for various industries? The answer is sugars. In the bioeconomy most processes rely on sugar and therefore we decided to convert the methanol into glycogen, the storage molecule of sugar in bacteria. This contributes to making the bioindustry independent of plants and instead rely on renewable methanol.


How We Improved a Previously Existing BioBrick

Enhancing the glycogen accumulation in Escherichia coli is an important aspect of our project. In this context we developed our glycogen synthesis operon (glgCAB). It is a composite part that combines all 3 glycogen formation enzymes. The ADP-glucose pyrophophorylase (GlgC) forms ADP-glucose from ATP and glucose-1-phosphate, the glycogen synthase (GlgA) elongates α-1,4-linked chains and the branching enzyme (GlgB) catalyzes the formation of α-1,6-linked branches. The GlgC is based on the part [http://parts.igem.org/Part:BBa_K118016 BBa_K118016] from Team Edinburgh 2008, but the RBS B0034 was added to the existing Biobrick. Since we could prove that strains expressing our polycistronic construct accumulate more glycogen than strains just expressing glgC, this part is an extension and improvement of the existing Part BBa_K118016.


The construct was confirmed by sequencing. The expression of all three enzymes GlgC, GlgA and GlgB was tested in BL21 Gold (DE3) strains containing BBa_K1585321 in a pSB1A30 expression vector.


Aachen glgCAB for registry.png
SDS-PAGE of glgCAB in pSB1A30
glgCAB in pSB1A30 was expressed in BL21 Gold (DE3) strains and IPTG induced. The small arrows indicates the expected bands for all three enzymes . The BL21 Gold (DE3) wild type was used as the negative control.

The combined functionality was characterized by iodine staining (see picture below). It was performed with Lugol's iodine which dyes glycogen in a brownish color. If more glycogen is present, the color of stainend cultures is darker. The darker staining of BL21 Gold (DE3) transformants of BBa_K1585321 indicates considerably more glycogen accumulations compared to the wild type.

Aachen glgCAB , WT v2.png
Iodine staining BL21 Gold (DE3) + glgCAB vs. wild type
Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) + glgCAB stained distinctly darker than the BL21 Gold (DE3) wild type.

References

  1. ↑ Bogorad IW, Chen CT, Theisen MK, Wu TY, Schlenz AR, Lam AT, Liao JC. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15928-33. doi: 10.1073/pnas.1413470111. Epub 2014 Oct 29. PubMed PMID: 25355907; PubMed Central PMCID: PMC4234558.
  2. ↑ http://www.sunfire.de/en/

▲
Contact
CC-BY-SA iGEM Aachen 2015