Difference between revisions of "Team:Marburg/CDI"

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<h1 >Background</h1>
 
<h1 >Background</h1>
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<p>
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Cdi (contact dependent inhibition) is a naturally occurring system in different strains of bacteria to deliver toxic subunits into target cells [1]. The Cdi+ cells inhibit the growth of the target cells [2] thus gain a growth advantage. The Cdi system is organized in an operon consisting of three genes: <br>
 +
• CdiB is a pore forming protein with a β barrel structure and might be needed in secretion and assembly of CdiA[3]. <br>
 +
• CdiA is the toxin delivering protein. The C-terminus (CT) of CdiA acts as toxic domain and got various orphan CdiA-CT units in behind[5].<br>
 +
• The third gene is CdiI, the inhibitor to protect the cells against their own toxins. <br>
 +
The structure of this operon differs from organism to organism and similar systems are found in various proteobacteria groups. In general, these operons consist of CdiB followed by CdiA including the first CdiA-CT behind a motif coding for the amino acids “VENN” followed by the third gene, the CdiI. Lastly, behind CdiI there were found several orphan Cdi-CTs ending with a resembling CdiI coding sequence. The current hypothesis is that the Cdi-CT domain encoding for the toxic subunit can be exchanged under currently unknown conditions. Due to the specific binding between each CdiI and the corresponding Cdi-CT there has to be one particular CdiI for each Cdi-CT. <br>
 +
The natural system is only expressed in logarithmic growth state of E.coli while growth inhibition can occur in log-phase as well as in stationary phase. The only requirement for a functional Cdi system is metabolic activity of the target proteobacteria, since most of the toxin subunits target the membrane with pore forming proteins or the metabolism with different nucleases. <br>
 +
After direct cell to cell contact the CDI operon will be transcribed and translated. CdiB presumably plays a role CdiA assembly and the widespread opinion is that the CdiA-Ct behind be VENN motive is not functional until secretion, cleavage and transport into the victim cell. After the pore in the Membrane of the host cell is formed by CdiB the CdiA delivery stick can be secreted and is exposed on the outer membrane. By getting in contact with the target cell’s BamA receptor in the outer membrane the toxic domain is cleaved off of CdiA and is delivered via ArcB transporter to the cytoplasm functioning as for instance nuclease [6, 8]. The transport mechanism of the toxin through the inner and outer membrane is still unclear [4]. If E.coli cells with the same CDi system get into contact while exposing CdiA on their surface the toxic subunit is nonetheless transported into the cytoplasm but gets inactivated by CdiI[9].
 +
Read more about the used toxic subunits and their corresponding growth inhibition in our Results. <br>
 +
 
 +
To conclude the CdiA/CdiB mediated growth inhibition system is a specific transport system for low Dalton proteins and could be developed to a great tool for synthetic biology to manipulate other proteobacteria without engineering them.
 +
 
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Revision as of 09:44, 18 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 E.coli system to create and establish a niche in the human gut microbiome targeting proteobacteria.
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 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.
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.

Figure 1:
The ability of CDI+ cells to inhibit the growth of CDI- target cells was tested in an inhibition assay. The target and inhibitor cells are mixed and grown for 22 hours in exponential phase. Constant conditions are accomplished by regular dilution using fresh medium.
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.
Figure 2:
In addition to flow-cytometry we wanted to observe the growth inhibitory effect on a cellular level. Cells were grown on agar pads and pictures were taken using fluorescence microscopy. The images visually confirm the results of the measurements obtained by flow cytometry.
Figure 3:

Outlook

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.

Background

Cdi (contact dependent inhibition) is a naturally occurring system in different strains of bacteria to deliver toxic subunits into target cells [1]. The Cdi+ cells inhibit the growth of the target cells [2] thus gain a growth advantage. The Cdi system is organized in an operon consisting of three genes:
• CdiB is a pore forming protein with a β barrel structure and might be needed in secretion and assembly of CdiA[3].
• CdiA is the toxin delivering protein. The C-terminus (CT) of CdiA acts as toxic domain and got various orphan CdiA-CT units in behind[5].
• The third gene is CdiI, the inhibitor to protect the cells against their own toxins.
The structure of this operon differs from organism to organism and similar systems are found in various proteobacteria groups. In general, these operons consist of CdiB followed by CdiA including the first CdiA-CT behind a motif coding for the amino acids “VENN” followed by the third gene, the CdiI. Lastly, behind CdiI there were found several orphan Cdi-CTs ending with a resembling CdiI coding sequence. The current hypothesis is that the Cdi-CT domain encoding for the toxic subunit can be exchanged under currently unknown conditions. Due to the specific binding between each CdiI and the corresponding Cdi-CT there has to be one particular CdiI for each Cdi-CT.
The natural system is only expressed in logarithmic growth state of E.coli while growth inhibition can occur in log-phase as well as in stationary phase. The only requirement for a functional Cdi system is metabolic activity of the target proteobacteria, since most of the toxin subunits target the membrane with pore forming proteins or the metabolism with different nucleases.
After direct cell to cell contact the CDI operon will be transcribed and translated. CdiB presumably plays a role CdiA assembly and the widespread opinion is that the CdiA-Ct behind be VENN motive is not functional until secretion, cleavage and transport into the victim cell. After the pore in the Membrane of the host cell is formed by CdiB the CdiA delivery stick can be secreted and is exposed on the outer membrane. By getting in contact with the target cell’s BamA receptor in the outer membrane the toxic domain is cleaved off of CdiA and is delivered via ArcB transporter to the cytoplasm functioning as for instance nuclease [6, 8]. The transport mechanism of the toxin through the inner and outer membrane is still unclear [4]. If E.coli cells with the same CDi system get into contact while exposing CdiA on their surface the toxic subunit is nonetheless transported into the cytoplasm but gets inactivated by CdiI[9]. Read more about the used toxic subunits and their corresponding growth inhibition in our Results.
To conclude the CdiA/CdiB mediated growth inhibition system is a specific transport system for low Dalton proteins and could be developed to a great tool for synthetic biology to manipulate other proteobacteria without engineering them.

iGEM Marburg - ZSM Karl-von-Frisch-Straße 16, D - 35043 Marburg