Difference between revisions of "Team:Marburg/CDI"
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<h1 >Project Design</h1> | <h1 >Project Design</h1> | ||
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| − | We have designed an <k>E.coli</> system to create and establish a niche in the human gut microbiome targeting proteobacteria. <br> | + | We have designed an <k>E.coli</k> system to create and establish a niche in the human gut microbiome targeting proteobacteria. <br> |
Therefore we created different constructs using the contact dependent growth inhibition system (CDI). This system inhibits growth of proteobacteria due to contact between CdiA and the receptor Protein BamA by which the inhibiting subdomain from CdiA is transported into the cytoplasm of the target cell. The Cdi cells are protected by the CdiI protein, which disables the inhibitory C-terminus of CdiA. This subdomain targets different important structures of proteobacteria for instance the membrane or DNA. To do so we chose a system with an inducible T5 Promoter and lac operator sides to control the expression of our growth inhibition system. To reduce leaky expression we cloned additional lacI controlled by BBa B0034 promoter into all our constructs to make it available for every strain. We decided to transform this construct into G10 Hicontrol strain, which is characterized by multiple lacI coding sequences. In a next step we decided to change from the high copy plasmid pSB1C to the low copy plasmid backbone pSB4C5 in order to reduce the metabolic burden caused by the large CdiA protein (300 kDa). <br> | Therefore we created different constructs using the contact dependent growth inhibition system (CDI). This system inhibits growth of proteobacteria due to contact between CdiA and the receptor Protein BamA by which the inhibiting subdomain from CdiA is transported into the cytoplasm of the target cell. The Cdi cells are protected by the CdiI protein, which disables the inhibitory C-terminus of CdiA. This subdomain targets different important structures of proteobacteria for instance the membrane or DNA. To do so we chose a system with an inducible T5 Promoter and lac operator sides to control the expression of our growth inhibition system. To reduce leaky expression we cloned additional lacI controlled by BBa B0034 promoter into all our constructs to make it available for every strain. We decided to transform this construct into G10 Hicontrol strain, which is characterized by multiple lacI coding sequences. In a next step we decided to change from the high copy plasmid pSB1C to the low copy plasmid backbone pSB4C5 in order to reduce the metabolic burden caused by the large CdiA protein (300 kDa). <br> | ||
| − | For further experiments we cloned a red fluorescent protein into two constructs. In the first it’s controlled by an inducible promoter while in the second it’s controlled by a constitutive promoter, which enables us to distinguish between E.coli with and without the Cdi system. For our target cells we used G10 Hicontrol as well and additionally, we transformed this strain with the interlab study’s construct BBa_K1650001 carrying green fluorescent protein in order to highlight them even more. <br> | + | For further experiments we cloned a red fluorescent protein into two constructs. In the first it’s controlled by an inducible promoter while in the second it’s controlled by a constitutive promoter, which enables us to distinguish between <k>E.coli</k> with and without the Cdi system. For our target cells we used G10 Hicontrol as well and additionally, we transformed this strain with the interlab study’s construct BBa_K1650001 carrying green fluorescent protein in order to highlight them even more. <br> |
For further applications we want to use the Cdi system to cut off a part of natural gut proteobacteria. In the future we are aiming to implement metabolic pathways in our cells carrying the CDI system which would enable us to produce beneficial compounds after creating a niche inside the human gut. <br> | For further applications we want to use the Cdi system to cut off a part of natural gut proteobacteria. In the future we are aiming to implement metabolic pathways in our cells carrying the CDI system which would enable us to produce beneficial compounds after creating a niche inside the human gut. <br> | ||
Revision as of 20:20, 17 September 2015
Aim
Overall, 842 million people suffer from malnutrition and 62 million deaths are related to it, annually. There is increasing evidence that the gut microbiome has a big influence on digestion and therefore on uptake of nutrients. We want to take advantage of therapeutic bacteria to establish a modified and beneficial sub-population in the human intestines. To accomplish this, we use the contact dependent inhibition system to cut off part of the natural gut population of proteobacteria whose higher appearance occurs in overweight people. Thereby, we want to create a niche for this synthetic sub-population.
Project Design
We have designed an
Therefore we created different constructs using the contact dependent growth inhibition system (CDI). This system inhibits growth of proteobacteria due to contact between CdiA and the receptor Protein BamA by which the inhibiting subdomain from CdiA is transported into the cytoplasm of the target cell. The Cdi cells are protected by the CdiI protein, which disables the inhibitory C-terminus of CdiA. This subdomain targets different important structures of proteobacteria for instance the membrane or DNA. To do so we chose a system with an inducible T5 Promoter and lac operator sides to control the expression of our growth inhibition system. To reduce leaky expression we cloned additional lacI controlled by BBa B0034 promoter into all our constructs to make it available for every strain. We decided to transform this construct into G10 Hicontrol strain, which is characterized by multiple lacI coding sequences. In a next step we decided to change from the high copy plasmid pSB1C to the low copy plasmid backbone pSB4C5 in order to reduce the metabolic burden caused by the large CdiA protein (300 kDa).
For further experiments we cloned a red fluorescent protein into two constructs. In the first it’s controlled by an inducible promoter while in the second it’s controlled by a constitutive promoter, which enables us to distinguish between
For further applications we want to use the Cdi system to cut off a part of natural gut proteobacteria. In the future we are aiming to implement metabolic pathways in our cells carrying the CDI system which would enable us to produce beneficial compounds after creating a niche inside the human gut.
Results
We constructed the CDI-system functionally in our E. coli lab strain and are able to induce the expression using IPTG. Proper functioning has been proven by co-cultivation of CDI+ and CDI- cells in which the change of ratio over time has been observed. Additionally we designed and built a functional prototype and showed that the CDI-cells can also be used when filled in a capsule.
We used construct BBa_K1650001, which was utilized for the Interlab study for engineering of target cells as well as construct BBa_B0007 for the engineering of inhibitor cells. Both plasmids are transformed into G10 Hi-control, a strain that expresses 60 fold LacI to avoid leaky expression.
If grown in separate wells of a 24 well plate, the CDI cells grow slightly slower due to metabolic burden caused by the huge proteins of the CDI operon.
The percentage of RFP labelled CDI+ inhibitor and of GFP labelled CDI- cells is measured using a flow cytometer. In this experiment we compared the change in CDI+/CDI- -ratio upon IPTG induction. If expression of the CDI-operon is induced a distinct increase of the fraction of the CDI+ cells occurs. This increase originates in growth inhibition of the target cells. So the growth disadvantage caused by metabolic burden is compensated by the ability to actively decrease the growth rate of rivals.
Outlook
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Background
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua.
