Team:TU Eindhoven/ZAP

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iGEM Team TU Eindhoven 2014

Zwitterionic Antifouling Protein

Another approach concerning the encapsulation of bacterial cells is to genetically engineer the cells in a way that enables them to produce their own coating. Therefore, we have designed a Zwitterionic Antifouling Protein (ZAP). A zwitterionic polymer can be displayed by an outer membrane protein on the cell’s outer membrane. In order to evade the immune system, a coating of these Zwitterionic Antifouling Proteins needs to be produced by the cell. Roughly, the zwitterionic encapsulation is the result of zwitterionic interactions of polymers consisting of mainly Glutamic Acid (negatively charged) and Lysine (positively charged). We have chosen for these amino acids due to the demonstrated antifouling properties of the combination of these negatively and positively charged amino acids. Sequences of repeats of these specific amino acids will interact with each other and are able to form a network which is impervious for antibodies. [1]

Plasmid design

First of all, we have designed a polymer chain consisting of mainly Glutamic Acid and Lysine. This polymer chain needs to be expressed onto the outside of the cell, but still has to be attached to its membrane. Therefore, we have incorporated the sequence for the zwitterionic polymer into the display part of the display protein the Penn 2012 team has used. This is the original Ice Nucelation Protein and was also used for COMPy. After quite some waiting on the synthesized antifouling sequence- or insert – and labor in the BioLab, we have successfully obtained and transformed the plasmid shown in figure 1. The protocols used in order to assemble this plasmids can be found here.

Evaluation of expression

ZAP has been designed with a HA-tag at the end of the zwitterionic sequence. To show whether or not the protein is expressed the bacteria are labeled with fluorescent anti-HA antibody. The antibodies were added to the bacteria after protein expression in two concentrations, 316 and 100 nM. After one hour of incubation the cells were washed and analyzed with FACS.

These results clearly shows that ZAP is expressed (figure 2). It is remarkable that there are quite a few cells with a lower level fluorescence.

Future prospects

ZAP has been designed with a HA-tag at the end of the zwitterionic sequence. To show whether or not the protein is expressed the bacteria are labeled with fluorescent anti-HA antibody. The antibodies were added to the bacteria after protein expression in two concentrations, 316 and 100 nM. After one hour of incubation the cells were washed and analyzed with FACS.

These results clearly shows that ZAP is expressed (figure 2). It is remarkable that there are quite a few cells with a lower level fluorescence.

Bibliography

[1] Yang, Q., Wang, L., Lin, W., Ma, G., Yuan, J., & Chen, S. (2014). Development of nonfouling polypeptides with uniform alternating charges by polycondensation of the covalently bonded dimer of glutamic acid and lysine. Journal of Materials Chemistry B, 2, 577-584.

iGEM Team TU Eindhoven 2014

@@ Line 126: / Line 126: @@
                  <div class="col_700_2 float_r">
-                   <h2>Zwitterionic Antifouling Approach</h2>
+                   <h2>Zwitterionic Antifouling Protein</h2>
-                   <p>A side project of this year’s TU Eindhoven iGEM Project is the Zwitterionic Encapsulation of a bacterial cell. The ultimate goal is however the same; create an antifouling encapsulation in order to evade the immune system. The difference is, however, that this approach encompasses a fully assembled system. Thus no alterations are possible as is the case with the click reaction.
+                   <p>Another approach concerning the encapsulation of bacterial cells is to genetically engineer the cells in a way that enables them to produce their own coating. Therefore, we have designed a Zwitterionic Antifouling Protein (ZAP). A zwitterionic polymer can be displayed by an outer membrane protein on the cell’s outer membrane. In order to evade the immune system, a coating of these Zwitterionic Antifouling Proteins needs to be produced by the cell. Roughly, the zwitterionic encapsulation is the result of zwitterionic interactions of polymers consisting of mainly Glutamic Acid (negatively charged) and Lysine (positively charged). We have chosen for these amino acids due to the demonstrated antifouling properties of the combination of these negatively and positively charged amino acids. Sequences of repeats of these specific amino acids will interact with each other and are able to form a network which is impervious for antibodies. [1]
-Roughly, the TU Eindhoven’s zwitterionic encapsulation is the result of zwitterionic interactions of polymers consisting of mainly Glutamic Acid (negatively charged) and Lysine (positively charged). We have chosen for these amino acids due to the demonstrated antifouling properties of the combination of these negatively and positively charged amino acids. [1]
 </p>
+                   <h3>Plasmid design</h3>
-                   <h3>Approach</h3>
 <p>
-The idea is therefore a polymer chain consisting of mainly Glutamic Acid and Lysine, bound to an anchor in order to facilitate the encapsulation around the bacterial cell. Penn iGEM team 2012 used an anchor which was able to bear a mCherry fluorescent group. Thus, the general idea is a bacterial cell littered with transmembrane anchors provided with a polymer chain comprising Glutamic Acids and Lysines. The amino acids interact with each other in order to form a network which is impervious for antibodies.
+First of all, we have designed a polymer chain consisting of mainly Glutamic Acid and Lysine. This polymer chain needs to be expressed onto the outside of the cell, but still has to be attached to its membrane. Therefore, we have incorporated the sequence for the zwitterionic polymer into the display part of the display protein the Penn 2012 team  has used. This is the original Ice Nucelation Protein and was also used for COMPy. After quite some waiting on the synthesized antifouling sequence- or insert – and labor in the BioLab, we have successfully obtained and transformed the plasmid shown in figure 1. The protocols used in order to assemble this plasmids can be found here.
+</p>
+<h3>Evaluation of expression</h3>
+<p>
+ZAP has been designed with a HA-tag at the end of the zwitterionic sequence.  To show whether or not the protein is expressed the bacteria are labeled with fluorescent anti-HA antibody. The antibodies were added to the bacteria after protein expression in two concentrations, 316 and 100 nM. After one hour of incubation the cells were washed and analyzed with FACS. <br><br>
+These results clearly shows that ZAP is expressed (figure 2). It is remarkable that there are quite a few cells with a lower level fluorescence.
+<h3>Future prospects</h3>
+<p>
+ZAP has been designed with a HA-tag at the end of the zwitterionic sequence.  To show whether or not the protein is expressed the bacteria are labeled with fluorescent anti-HA antibody. The antibodies were added to the bacteria after protein expression in two concentrations, 316 and 100 nM. After one hour of incubation the cells were washed and analyzed with FACS. <br><br>
+These results clearly shows that ZAP is expressed (figure 2). It is remarkable that there are quite a few cells with a lower level fluorescence.
 </p>
 <h4>Bibliography</h4>
 <p>[1] Yang, Q., Wang, L., Lin, W., Ma, G., Yuan, J., & Chen, S. (2014). Development of nonfouling polypeptides with uniform alternating charges by polycondensation of the covalently bonded dimer of glutamic acid and lysine. Journal of Materials Chemistry B, 2, 577-584.