Description

Introduction

Our project is called SPACE-P (Structural Phage Assisted Continuous Evolution of Proteins) and is part of the Synthetic Biology field. It focuses on accelerating the process of developing protein binding partners, which is required in pharmaceutical research as well as biotechnology and many other fields of science. Thus far, phage display is the most commonly used method for the discovery of protein binding partners. In this method a large library of potential binding partners is created where the strongest candidates are selected for further affinity testing. This process is very time consuming, cost intensive, requires human intervention steps, and is limited to the size of the given library. Our platform will make it possible to start with a single molecule, transform it by mutation and selection pressure to improve the binding affinity towards a target protein. This method is not only easy to implement, utilizing simple organisms and devices, but also significantly faster and cheaper than currently used tools.

Subprojects

To establish our idea we divided the whole work into subprojects which were done simultaniously. These subprojects would then be combined later. Troubleshooting issues is much easier with this approach and more efficient compared to a step by step approach where you start the next subproject only when you succeed with the previous ones. The subprojects we worked on are the following:

• 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

Folding study of target protein

As a proof of principle we wanted to fit an affibody to the epitope of a target protein. The target we chose for our studies is a human membrane protein called human epidermal growth factor receptor 2 (HER2). To use a human protein and membrane protein is challenging since the codon usage, post-translational modifications, disulfide bridges and membrane parts have to be taken into account. To deal with this challenges we decided for a small extracellular domain that carries no post-translational modifications and disulfide bridges. This part of HER2 is involved in ligand binding and therefore is an interesting drug target. Afterwards we harmonized the codon usage to E. coli. Finally we compared the protein structure expressed from E. coli to the expected one from literature. To do so we cloned the fragment into an expression vector with a tag, purified it with a column and analyzed it with circular dichroism spectroscopy.

Structure analysis of our targets and their interactions

Having the crystallized structure of the extracellular part of HER2 and the affibody ZHER2 binding to it (PDB-ID: 3MZW), we are able to perform further investigations on our targets and their interactions. Those analysis should give us a presentiment of possibilities for the directed evolution and therefore provide a benchmark for the choice of further possible targets. The following analysis are performed:

• Structure check of HER2
• Calculation of interfacial residues of HER2 and its bound affibody
• Calculation of electrostatic interactions in the interface
• Conservation study of HER2
• Visualization of the b-factor for the affibody ZHER2

Investigation of P3 threshold for E. coli resistance

The protein III (P3) from M13 plays a key role in our concept. The deficiency of it in the M13 phage we used can be compensated by the modified E. coli. However it is known that the expression of P3 in E. coli before the infection with M13 leads to the resistance of the bacterium to the phage. This was our motivation to find the threshold of resistance. To find it we measured the amount of infected bacteria cells in the lagoon depending on the expression rate of P3. The P3 expression rate was detected by the co-expression of a fluorescent protein while the amount of infected cells was determined with the help of a blue-white screening. In this assay the cells that carry a phage are blue on X-Gal plates due to the complementation of the lacZ gene by the phages.

Conversion of BACTH into an iGEM standard and analysis of function

With the combination of PACE and BACTH for our project, we are able to investigate the affinity of any two proteins of interest. Since this assay was lacking from the registry, it became evident that we could create it, built up from biobricks. So we went to work ordering T25 and T18 as biobricks as well as leucine zippers as our “proteins of interest”, positive controls and proof of principle that this assay is biobrick compatible. With a fusion ligation of the leucine zippers to their respective T25 or T18, the build up began. This was followed by a non-fusion ligation in order to piece the entire construct together. Lastly the construct was inserted downstream of a lacZ promoter (pLac), expressed in a cya- strain in order to obtain a read out on X-gal plates. In this assay the cells carrying the positive control and successful protein protein interaction will appear blue due to the activation of lacZ from newly synthesized cAMP.

Set up of flow system

During the initial conception of the project different parameters for the cultivation system were described. All of them showed different media compositions as well as different volumes for the bioreactor and lagoons. This led us to the idea of optimizing and adapting the whole system to our specific needs. Our main focus was on making the system more efficient by using less recourses and producing less waste.

The paper by Esvelt et al. (2011) describes a variety of PACE systems, which follow the setup in figure 1. For continuous cultivation the stirred-tank reactor was diluted with fresh medium. The same amount of medium containing E. coli was pumped out of the reactor. The growth of the bacteria was controlled by the dilution rates (1 – 2.5 h^-1). However the smaller reactor (lagoon, 4) had a dilution of 1 h^-1. Therefore the redundant liquid from the continuous stirred-tank reactor (CSR) was separated into a waste container (3). The lagoon was infected with phages which aimed to reproduce inside the E. coli. The final goal of the experiment was to induce an evolutionary process of the phages inside the lagoon.

After accomplishing a more efficient system the next step in our work was to find out how our organism grows within the bioreactor and if the plasmid is stable within the organism as well as inducible.

Figure 1 - Setup of the PACE systems.

Biobrick assembly

The individual protein coding sequences used for the various subprojects were encoded as separate Biobrick parts with the necessary prefix and suffix as per iGEM standards. These biobricks were initially synthesized by IDT and later encoded in the iGEM standard plasmid backbone pSB1C3. Following which, the parts were sequenced to confirm the proper insertions. The individual subproject utilization of the Biobrick parts also adds to characterizing each part.

To the top!