Team:TU Eindhoven/Achievements/Future Prospects

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iGEM Team TU Eindhoven 2014

Future Prospects

Figure 1. Schematic overview of the Rolling
Circle Amplification principle. A short circular
template is continuously transcribed to form a
long piece of single strand DNA.

Click Coli

We have successfully developed the Click Coli, which is a versatile tool when it comes to engineering the outer membranes of E. coli. However, there are still some aspects that remain unknown and require more research. These aspects are discussed below.

Proliferation

During the characterization of the Click Coli system, the genetically engineered E. coli bacteria have always been kept at 4 degrees Celsius. At this temperature proliferation is highly unfavorable. We kept the bacteria on purpose under this condition in order to inhibit cell division. However, in many possible applications of Click Coli this condition cannot be met. Further research is required in order to gain insight into the effects of proliferation on the Click Coli system. For instance, it would be valuable to know how the membrane proteins would be distributed amongst the daughter cells.

Quantification of Clickable Outer Membrane Proteins

So far, only qualitative data on the amount of Clickable Outer Membrane Proteins has been obtained by FACS. The FACS results only show the level of fluorescence per cell, which is an indirect way of evaluating the amount of expressed Clickable Outer Membrane Proteins. The amount of expressed protein has a great impact on the properties of the coating, since it determines its density. A more precise quantification of the total amount would be useful in the refinement of a coating. For instance, determining the amount of DBCO functionalized molecules that need to be added to the bacteria in order to use all available binding sites on the membrane proteins.

Permeability of coating

If the E. coli bacteria are coated, this has quite possibly an effect on the uptake and secretion of molecules. However, the properties of the coating are completely dependent on the kind of molecules this coating is composed of. Therefore, in order to obtain information on which types of molecules are still able to cross the cell membrane, the permeability of each specific coating should be evaluated.

Applications of Click Coli

The properties and effects of the genetically engineered bacteria that are modified with use of the Click Coli system are completely dependent on the type of molecules that are attached to the outer membrane, as well as additional genetically engineered functions. We see a lot of opportunities for the Click Coli system: some future application scenarios are described in our Synenergene project . Apart from the formation of a functional coating, Click Coli could also be used to immobilize bacteria. This would be convenient for further application in bioreactors or advantageous bio based filters.

Rolling Circle Amplification on cells

As was mentioned in the introduction of Rolling Circle Amplifcation there are many possible reasons why it is useful to attach DNA to the outside of cell membranes. The obvious example would be aptamers which display similar recognition and diversity as antibodies. They provide an advantage in the form of the ability to bind to toxins to which a normal immune response is not triggered. [1] This means these aptamers could be used if combined to signaling proteins to help bacteria sense their environment even better than before. A system consisting of one generic signal-relay protein and a set of clickable aptamers could be a great start for many functional cells with easily changeable ligands. It is also possible to create DNA with protein like functionalities in order to create so called DNA enzymes. These enzymes have been created to catalyze a diverse range of reactions, including RNA cleavage, DNA cleavage, RNA ligation, DNA ligation, deglycosylation, and other enzymatic activities as peroxidases. [2] Since DNA can be made to function as an enzyme we envision a system in which a DNA based enzyme could work as a pre-processor before a potentially harmful metabolite enters the bacteria. The DNA enzyme coating surrounding the cell can break down toxins so that the cell can utilize them. The advantage of using DNA over proteins clicked to the cell is the same as for aptamers, DNA has proven to be much more stable than proteins. Lastly, DNA also shows promising capabilities as a designer material. Since DNA can be made to form self-assembling structures whose function and form can be determined in advance, it is possible to design many interesting shapes. [3] The field of DNA engineering is just starting out, but already many applications have been developed to make use of DNA’s unique ability to form links to complementary strands. [4] Also it has been shown that DNA can be used to form hydrogels, which could potentially mean that DNA can be used to coat cells in an antifouling coating which has therapeutic use. [5]

Bibliography

[1] Navani, N., & Li, Y. (2006). Nucleic acid aptamers and enzymes as sensors.Current Opinion in Chemical Biology,10(3), 272-281.

[2] Liu, J., Cao, Z., & Lu, Y. (2009). Functional Nucleic Acid Sensors. Chemical Reviews,109(5), 1948-1998.

[3] Seeman, N. C. (2003). DNA in a material world. Nature, 421(6921), 427-431.

[4] Cadnano. (n.d.). cadnano. Retrieved October 14, 2014, from http://cadnano.org/

[5] Lee, J. B., Wu, M., Luo, D., Long, R., Chen, L., Rice, E. J., et al. (2012). A mechanical metamaterial made from a DNA hydrogel. Nature Nanotechnology,7(12), 816-820.

iGEM Team TU Eindhoven 2014

@@ Line 126: / Line 126: @@
                  <div class="col_700_2 float_r">
-                   <h2>Rolling Circle Amplification</h2>
+                   <h2>Future Prospects</h2>
 <figure style="float:right;margin-right:0;">
@@ Line 132: / Line 132: @@
 <figcaption style="font-size:18px;color:#CCCCCC;">Figure 1. Schematic overview of the Rolling<br> Circle Amplification principle. A short circular <br> template is continuously transcribed to form a <br> long piece of single strand DNA. </figcaption>
 </figure>
+<h3>Click Coli </h3>
-                   <p>One way to use the Click Coli system developed by iGEM Eindhoven 2014 is to functionalize the outside of the bacterial cell membrane with DNA molecules. This offers many exciting possibilities for applications. For example, Bertozzi et al. [1] showed that DNA-bound to the outside of cells could be used for 3-dimensional tissue engineering. This technique would allow a vast array of applications where two or more cell types have to communicate with each other to be more finely controlled.
+                   <p>We have successfully developed the Click Coli, which is a versatile tool when it comes to engineering the outer membranes of E. coli. However, there are still some aspects that remain unknown and require more research. These aspects are discussed below.</p>
-<br><br>
+<h4>Proliferation</h4>
-Another application is covering the membrane with functional aptamers, which can be used for targeting specific molecules or diseases [2-5]. Also, Lee et al. showed that DNA can be used to form a hydrogel like material, which has potentially interesting properties when coupled to a cell membrane [6]. All these functionalities have in common that they are almost always synthesized using so called Rolling Circle Amplification. The Eindhoven iGEM 2014 tries to use Rolling Circle Amplification to create a functional coating around the bacterial cell using the Click Coli system and specifically engineered DNA templates.
+<p>During the characterization of the Click Coli system, the genetically engineered E. coli bacteria have always been kept at 4 degrees Celsius. At this temperature proliferation is highly unfavorable. We kept the bacteria on purpose under this condition in order to inhibit cell division. However, in many possible applications of Click Coli this condition cannot be met. Further research is required in order to gain insight into the effects of proliferation on the Click Coli system. For instance, it would be valuable to know how the membrane proteins would be distributed amongst the daughter cells. </p>
-<br><br>
+<h4>Quantification of Clickable Outer Membrane Proteins</h4>
-Rolling Circle Amplification creates a long strand of single strand DNA by “rolling” a circular template along that is used for amplification. Because a circular template is used the strands can, theoretically, be as long as needed. But they will also contain many repeats of the same template. We demonstrated that this can be used to greatly increase the number of binding sites on the bacterial cell membrane.
+<p>So far, only qualitative data on the amount of Clickable Outer Membrane Proteins has been obtained by FACS. The FACS results only show the level of fluorescence per cell, which is an indirect way of evaluating the amount of expressed Clickable Outer Membrane Proteins. The amount of expressed protein has a great impact on the properties of the coating, since it determines its density. A more precise quantification of the total amount would be useful in the refinement of a coating. For instance, determining the amount of DBCO functionalized molecules that need to be added to the bacteria in order to use all available binding sites on the membrane proteins. </p>
+<h4>Permeability of coating</h4>
+<p>If the E. coli bacteria are coated, this has quite possibly an effect  on the uptake and secretion of molecules. However, the properties of the coating are completely dependent on the kind of molecules this coating is composed of. Therefore, in order to obtain information on which types of molecules are still able to cross the cell membrane, the permeability of each specific coating should be evaluated.</p>
+<h3>Applications of Click Coli</h3>
+<p>The properties and effects of the genetically engineered bacteria that are modified with use of the Click Coli system are completely dependent on the type of molecules that are attached to the outer membrane, as well as additional genetically engineered functions. We see a lot of opportunities for the Click Coli system: some future application scenarios are described in our Synenergene project . Apart from the formation of a functional coating, Click Coli could also be used to immobilize bacteria. This would be convenient for further application in bioreactors or advantageous bio based filters. </p>
+<h3>Rolling Circle Amplification on cells</h3>
+<p>As was mentioned in the introduction of Rolling Circle Amplifcation  there are many possible reasons why it is useful to attach DNA to the outside of cell membranes. The obvious example would be aptamers which display similar recognition and diversity as antibodies. They provide an advantage in the form of the ability to bind to toxins to which a normal immune response is not triggered. [1] This means these aptamers could be used if combined to signaling proteins to help bacteria sense their environment even better than before. A system consisting of one generic signal-relay protein and a set of clickable aptamers could be a great start for many functional cells with easily changeable ligands.
+It is also possible to create DNA with protein like functionalities in order to create so called DNA enzymes. These enzymes have been created to catalyze a diverse range of reactions, including RNA cleavage, DNA cleavage, RNA ligation, DNA ligation, deglycosylation, and other enzymatic activities as peroxidases. [2] Since DNA can be made to function as an enzyme we envision a system in which a DNA based enzyme could work as a pre-processor before a potentially harmful metabolite enters the bacteria. The DNA enzyme coating surrounding the cell can break down toxins so that the cell can utilize them. The advantage of using DNA over proteins clicked to the cell is the same as for aptamers, DNA has proven to be much more stable than proteins.
+Lastly, DNA also shows promising capabilities as a designer material. Since DNA can be made to form self-assembling structures whose function and form can be determined in advance, it is possible to design many interesting shapes. [3] The field of DNA engineering is just starting out, but already many applications have been developed to make use of DNA’s unique ability to form links to complementary strands. [4] Also it has been shown that DNA can be used to form hydrogels, which could potentially mean that DNA can be used to coat cells in an antifouling coating which has therapeutic use. [5]
 </p>
 <h4>Bibliography</h4>
-<p>[1] Gartner, Z. J., & Bertozzi, C. R. (2009). Programmed assembly of 3-dimensional microtissues with defined cellular connectivity. Proceedings of the National Academy of Sciences, 106(12), 4606-4610.
+<p>[1] Navani, N., & Li, Y. (2006). Nucleic acid aptamers and enzymes as sensors.Current Opinion in Chemical Biology,10(3), 272-281.
-<br><br>
-[2] Huang, Y., Cheng, X., Duan, N., Wu, S., Wang, Z., Wei, X., et al. (2015). Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1. Food Chemistry, 166(1), 623-629.
 <br><br>
-[3] Cha, T., Cho, S., Kim, Y., & Lee, J. (2014). Rapid aptasensor capable of simply diagnosing prostate cance. Biosensors and Bioelectronics, 62, 31-37.
+[2] Liu, J., Cao, Z., & Lu, Y. (2009). Functional Nucleic Acid Sensors. Chemical Reviews,109(5), 1948-1998.
 <br><br>
-[4] Chen, H., Hou, Y., Qi, F., Zhang, J., Koh, K., Shen, Z., et al. (2014). Detection of vascular endothelial growth factor based on rolling circle amplification as a means of signal enhancement in surface plasmon resonance. Biosensors and Bioelectronics,61, 83-87.
+[3] Seeman, N. C. (2003). DNA in a material world. Nature, 421(6921), 427-431.
 <br><br>
-[5] Hu, R., Zhang, X., Zhao, Z., Zhu, G., Chen, T., Fu, T. and Tan, W. (2014), DNA Nanoflowers for Multiplexed Cellular Imaging and Traceable Targeted Drug Delivery. Angew. Chem. Int. Ed., 53: 5821–5826.
+[4] Cadnano. (n.d.). cadnano. Retrieved October 14, 2014, from http://cadnano.org/
 <br><br>
-[6] Lee, J. B., Wu, M., Luo, D., Long, R., Chen, L., Rice, E. J., et al. (2012). A mechanical metamaterial made from a DNA hydrogel. Nature Nanotechnology,7(12), 816-820.
+[5] Lee, J. B., Wu, M., Luo, D., Long, R., Chen, L., Rice, E. J., et al. (2012). A mechanical metamaterial made from a DNA hydrogel. Nature Nanotechnology,7(12), 816-820.
 </p>