Conclusions

Our results for the different subprojects led us to the following conclusions:

• 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

In order to obtain a first presentiment of possibilities for the directed evolution several structure analysis steps were performed.

First of all the PDB structure of HER2 and its bound artificial affibody, which had been acquired by x-ray diffraction, was validated using the Ramachandran plot for verification of the dihedral angles and the experimental details as a fairly good resolution of 2.9 Å, a R-value of 0.208 and a R-free of 0.278. This ensures that it is a suitable structure for further structure investigations.

The conservation study of HER2 and the visualization of the B-factor of the affibody ZHER2 gave us further insights into the flexibility of both structures. Those analyses show that HER2 is rather conserved in the interfacial residues and that also the affibody is less flexible where it forms the interface with HER2.

The interaction area of both molecules is in general not very large, nevertheless they structurally fit to each other very well and they form 9 hydrogen bonds, resulting in a very strong binding. This was also suggested previously with the experimentally obtained dissociation constant K_D of 22 pM.

All in all, the findings suggest that an affibody, having a small and stable structure, is a suitable molecule for a directed evolution, since ZHER2's high-affinity binding has evolved during selection and affinity maturation. It finally demonstrates that high-affinity binding can be obtained by binding surface optimization and stability and does not necessarily require a large interface - just the right atoms at the right position. Reaching this in a low cost and low time intensive manner is our aim in SPACE-P.

Investigation of P3 threshold for E. coli resistance

Some difficulties were found with the expression of P3 and the resulting washout of the phages. The plasmid showed itself stable during the whole cultivation which makes the next step a cultivation without antibiotics possible and highly desirable.

Further experiments regarding the plasmid stability should be performed, as well as reducing the yeast extract from the medium by identifying the missing element in the medium. A special expression analysis has to be done on P3 in relation to the IPTG-concentration.

In order to ensure proper antibiotic denaturation the cultivated medium had to be sterilized for 20 minutes at 121 °C. Further experiments are necessary to test the need of antibiotics for plasmid stability in the system. Since the antibiotic resistance of the recombinant E. coli strand is based on chloramphenicol degradation..

Additionally, the synthesized enzyme is secreted into the cultivation medium. This might lead to a complete loss of antibiotic function and therefore allow plasmid free E. coli to reproduce. As a result, the plasmid free cells might accumulate inside the CSR. Thus it is necessary to compare the plasmid stability of antibiotic free and antibiotic enriched media. Accounting to the previous reasons the influence of the chloramphenicol on plasmid stability might be negligible. As a result other ways to support plasmid stability or antibiotic free systems might be used for further experiments.

Conversion of BACTH into an iGEM standard and analysis of function

Set up of flow system

The continuous cultivation and CFP expression analysis gave a first idea of the steps needed to perform in order to obtain a continuous evolutionary process. It was possible to prove that the continuous flow system works as expected and that the phages are able to infect the E. coli.

All in all, the initial experiments helped us to grasp the idea of how such experiments can look like and where they are headed. We could show that our flow system works with the phages and the waste could be reduced to a minimum.