Results

• Correct folding study of target protein
• Structure analysis of our targets and their interactions
• Investigation of P3 threshold for E. coli resistance
• Conversion of BACTH into an iGEM standard and analysis of function
• Set up of flow system

Correct folding study of target protein

Structure analysis of our targets and their interactions

Structure check of HER2

The Ramachandran plot shows the phi-psi torsion angles for all residues in the structure (except those at the chain termini). Glycine residues are separately identified by triangles as these are not restricted to the regions of the plot appropriate to the other sidechain types.

The colouring/shading on the plot represents regions with different favorability: the darkest areas (here shown in red) correspond to the "core" regions representing the most favourable combinations of phi-psi values.

Ideally, one would hope to have over 90% of the residues in these "core" regions. The percentage of residues in the "core" regions is one of the better guides to stereochemical quality.

Interactions of HER2 and its affibody

After definition of the interfaciual atoms, electrostatic interactions in the interface can be defined and visualized as shown in Figure 1.

A total number of 9 hydrogen bonds were identified between HER2 and its affibody. Those are listed below with their respective distances.

Conservation study of HER2

In order to get an impression about possible variabilities of the HER2 structure a conservation study of HER2 was performed using 11 structures from different organisms (Figure 2). The multiple sequence alignment which is required for the calculation can be seen here. Looking at the binding interface of HER2 and its affibody, we can state that the regions where both get into contact are rather conserved.

Figure 2 - HER2 conservation - calculated using 11 HER2 structures from different organisms.

Negative results:

Performing the rather automatic analysis of HER2 conservation by using all available HER2 structures gives a very large amount of structures to compare with.

This results in large alignment gaps and in an overall relatively low conservation without larger shade differences except for single amino acid (the whole Amino Acid Conservation Scores can be found here and the first lines of the color coded alignment can be seen here). Therefore the the sample is too large for a nice visualization and also the database structures might be biased.

In case of the affibody a conservation analysis could not be performed since it is an artificially engineered molecule. Therefore, in order to nevertheless get an impression about possible variabilities of the affibody structure an analysis of its cristallographic B-factors was performed.

Visualization of the B-factor for the affibody ZHER2

In crystallography the B-factor, also called temperature factor or "Debye-Waller factor", describes the displacement of an atom from its mean position in a crystal structure. The displacement may be the result of temperature-dependent atomic vibrations or static disorder in a crystal lattice. Static disorder means that some regions of the molecule may adopt different conformations in different copies of the molecule, each molecule's conformation being relatively stable. In the case of our affibody static disorder is not so probable, since it is a very small protein, designed to adopt a stable conformation.

Reflecting the disorder of an atom, the B-factor is therefore an indicator for flexibility caused by thermal motion.

As depicted in the following pictures the affibody has low B-factor values, meaning that it stays in a stable position without any larger fluctuations (indicated by the blue color). Only at the ends of the molecule a slight increase of the B-factor can be stated. This is normal and due to thermal motion, since the atoms have less interaction partners there, which can hold them on place. This stable position of the affibody suggests a high binding affinity at this position.

Affibody ZHER2 surface coloured by b-factor	Affibody ZHER2 structure coloured by b-factor	Affibody ZHER2 structure coloured by b-factor

Video

The following video shows the structure of the extracellular regions of HER2 with the affinity matured 3-helix affibody ZHER2 (PDB-ID: 3MZW) and focuses on their interaction, whereas hydrogen bonds are represented as dashed yellow lines and then the complete interacting interface is represented as surface, colored by atom type (N-blue, O-red).

Investigation of P3 threshold for E. coli resistance

To study the resistance that is build up by E. coli as response to P3 production, we constructed a plasmid that carries P3. The expression can be controlled by the inducible lac promoter. At the same time the reporter CFP is coexpressed (Figure 4).

Figure 4 - Final construct of pLac, P3, RBS and CFP. The induction of the promoter leads to the expression of P3 and the coexpression of CFP as a reporter.

The plasmid it was transformed into the F+, Δ(lacZ) E. coli strain after its construction. The F+ gives the M13 phages the possibility to infect the bacteria. Because of the lacZ deficiency the alpha-complementation can be carried out by the phages and the cells can be used for lacZ/blue-white screening. To have a very simple check for the functionality of the construct, we placed the induced cells on a UV table. A clear blue fluorescence could be seen, but not distinguished from the auto-fluorescence of the LB medium. Thus we performed the more sophisticated analysis of the induced cells with a 3D scan over a wide range of excitation and emission wavelengths. Results from Carl plus explanation The fluorescence is the parameter that corresponds to the P3 expression.

To get hands on the grade of resistance we used the blue-white screening that gives a ratio of infected (blue) to uninfected cells (white). Before we conducted the measurement in the reactor we tested if the blue-white screening works as assumed in simple reaction tubes (Figure 5) and on LB plates (Figure 6).

Figure 5 - Left: ER2738 cells induced with Xgal but without M13mp18. Right: Production of blue dye as a response to the infection with M13mp18. The phage performs the alpha-complementation of the damaged Β-galactosidase. The enzyme can then convert X-gal into the blue indigo dye.

Figure 6 - The plate was covered with top-agar which contained infected and uninfected cells. A continuous bacteria carpet developed and turned blue at the spots where infected cells grew. The bigger blue spots arose from condensate drops that allowed the phages to spread after the top agar was deposited.

Conversion of BACTH into an iGEM standard and analysis of function

Set up of flow system