Team:TU Eindhoven/ZAP

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 most important parts of this plasmid are, in order, the display protein, an alpha linker for flexibility, our Glutamic Acid- and Lysine-rich sequence, linker for flexibility and finally a HA-tag for immuno assays. The protocols used in order to assemble this plasmids by means of digestion and ligation can be found on the Protocol page.

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 antibodies. The antibodies were added to the bacteria after protein expression in a concentrations of 316 nM. After one hour of incubation the cells were washed and analyzed with FACS. These results clearly shows that ZAP is expressed (Figure 2) due to the shift of the graph to the right. This indicates protein expression and thus expression of ZAP. It is remarkable that there are quite a few cells with a lower level fluorescence.

Future prospects

Unfortunately, the process so far in designing and characterizing ZAP has been quite time-consuming. It was not possible to further investigate the antifouling properties of the E. coli that have expressed these Zwitterionic Antifouling Proteins before the deadline of the iGEM wikifreeze. Since there were no antifouling testing antibodies commercial available for the bacteria strain (BL21 DE3) – these are merely available for the cell’s lysate and not for living cells, no easy test could be performed on the ZAP producing cells. We suggest relocating the HA-tag in the protein structure to the place closest to the outside of the cell’s outer membrane. After expression of this variant of ZAP, fluorescently labelled Anti-HA anti-bodies could be added to the E. coli. The level of fluorescence determined by FACS would provide more insight in the antifouling properties of the ZAP coating.

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.