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
- Biobrick assembly
Correct folding study of target protein
We were able to observe the expression and folding of the part designed BBa_K1781001, which codes for the extracellular domain of the HER2 protein. The expression was observed in E. coli BL21 strain.
As observed from SDS-PAGE and a native PAGE we were able to identify our 22 kDa protein of interest. CD analysis on the protein revealed that, our protein of interest produced proper secondary structures as observed in the actual crystallised form of the protein.
The folding study of this part (HER2 extracellular domain coding region) is critical not only for the standard characterization of the coding region, but also for further downstream experiments. The proper folding of HER2 will enable the affibody protein encoded by the M13 phage to evolve to fit to HER2 and produce strong binding interaction. Hence, proper folding to its native in vivo structure will result in formation of affibody, which binds correctly to the extracellular domain of HER2. Improper folding of HER2 might lead to evolution of affibody protein that might not work when it is used to detect HER2 from human blood.
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 KD 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 of 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 minimal 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 for 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
The conversion of BACTH into an iGEM standard involved adding restriction sites which acted as prefixes (EcoRI and XbaI) and suffixes (SpeI and PstI site). The final construct also has the common prefixes and suffixes, thereby making it easier to use with other Biobricks.
The idea of creating an assay using BACTH system needs further validation. To act as a proof of study, we used leucine zipper sequences fusion-ligated with T18 and T25. If expressed properly, the leucine zippers would bind and the T18 and T25 domains of the adenylate cyclase enzyme would come closer and convert ATP to cAMP. But adenylate cyclase is produced in E.coli by default, so we chose to use an adenylate cyclase deficient strain to express the plasmid containing the fusion ligated T18 and T25 (BTH101). To induce the plasmid expression the T18 and T25 sequences were preceded by a lac promoter, which would get activated in presence of IPTG and when grown in an X-Gal plate should produce blue colour. But from the results obtained we had many white colonies along with the blue colonies. If blue colonies were produced that would mean that the T18 and T25 domains had come close enough to produce cAMP which in turn would mean that the proteins to which the domains were fused to, have really good binding. On the other hand the observance of white colonies indicated problems with transformation, or in the preparation of X-Gal plates.
The observance of blue colonies is to be followed up with further expression analysis for the respective proteins and other correlative binding assays. Once this procedure could be established, this could be used as an easy way of detecting protein-protein interactions.
Set up of flow system
The continuous cultivation and CFP expression analysis gave a first idea of the steps which need to perform for 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 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.
We created a separate section for our Biobricks. Further information can be found under Parts.