Our Best Basic PartWe present as best basic part the codon-optimized version of the cytochrome c protein that is able to convert silicon educts to organosilicon products.
Our Improved PartOur improved part, beta-lactamase_E102F, was designed to confirm the generative capabilities of our software package. Our algorithm has proven to be successful by engineering the enzyme to exhibit more than twice the activity of the wildtype protein.
Our Parts CollectionWe present the first self-contained in vivo evolution toolbox comprising 26 standardized BioBricks. At the heart of our toolbox is an innovative workflow for enzyme engineering, paving the way towards bio-production of novel compounds. It comprises promiscuous enzyme candidates (CYP1A2 and Cytochrome c) and selection-mediating accessory parts such as our riboswitch for detection of an organosilicon (BBa_K2398555).
Our RFCOur BBF RFC combines the methodical aspects of this year´s iGEM Team Heidelberg project, presenting a new golden-gate cloning standard for simple production of geneIII-deficient phages carrying any gene-of-interest and the first accessory plasmid construction kit for selection of the best phage library member on the one hand, while having our easy-to-use and low-cost bacteriophage based in vivo directed method PREDCEL (Phage-RElated DisContinous EvoLution) on the other hand. Together with our ODE-based modeling and computer simulations performed in context of AIGEM, our RFC opens up the principle of Phage-assisted continous evolution (PACE) to the scientific world, enabling every other iGEM team to perform directed evolution on their own without the highly challenging task of setting up a PACE apparatus.
Our Best Basic Part: Cytochrome C
As a next step, this part can be implemented in the directed evolution approach of phage-assisted continuous evolution (PACE) or in the phage-related discontinuous evolution (PREDCEL) approach to improve organosilicon synthesis by cytochrome engineering. For the characterization of this part, we conducted experiments for the production of organosilicons and therefore used a previously described triple mutant, already engineered by [Arnold et al., 2016]. According to the protocol, we obtained the matured cytochrome c and performed conversion experiments analyzed via GC-MS method.
The following figures show the results obtained with the previously engineered enzyme. First, we tested the enzyme on the conversion of the compound (1) with (3) to the final product of (5) (Fig. 1). The gas chromatogram shows one product peak which emerges at 9.2 min retention time. Analysis of the gas chromatogram indicates a conversion rate of >99% on the basis of the non-existence of the respective educt peak. As we performed GC-MS analysis, mass spectrometry data were also available which can be seen in Fig. 3 for the product peak that emerges at 9.2 minutes. It shows the fission of this product and can clearly be identified as our desired product (5). Furthermore, we performed experiments on the compound (1) which differs from compound (2) in its amino functional group. The product is eluted after 11.7 minutes retention time (Fig. 4) and also confirmed as the right product via mass spectrometry in Fig. 5. Worth mentioning, there is still educt (1) present which emerges at 6.9 minutes retention time, indicating incomplete conversion. Calculations showed a conversion rate of 47.5% for the product (4).As these results only show activity for a previously engineered cytochrome c, we are convinced that our part is still functional despite not as effective as the mutant. This part as our best basic part is intended as a platform for future teams to evolve their own cytochrome c for their specific target molecule.
Our Best Composite Part
BBa_K2398019: Theophylline Riboswitch - GeneIII for the Evolution of EnzymesOne of our main goals was to facilitate for the in vivo directed evolution of small molecule binding enzymes. The concept behind this idea is the use of riboswitches for the activation of geneIII. To prove our idea, we made use of a theophylline binding riboswitch in relation with the Enzyme CYP1A2, which catalyzes the reaction of caffeine to theophylline. As we could show that the principle of enzyme evolution with the help of riboswitches works, our best composite part is the theophylline riboswitch, followed by geneIII and flanked ba homology regions, which make it compatible with our cloning standard.
Our evolution circuit for cytochrome engineering works as follows: Bacteriophages infect bacterial cells by introducing their genome. The genome encodes a Selection Plasmid (SP) that contains the human CYP1A2 variant and all necessary components for virus propagation except geneIII. The Accessory Plasmid (AP) codes for geneIII driven by a Psp-tet promoter and contains a riboswitch, located between the promoter and geneIII, which regulates the expression rate of geneIII. The riboswitch on the AP is only active if theophylline reaches a certain concentration within the bacterium. If CYP1A2 is active, caffeine is converted to theophylline. Gradually the theophylline concentration increases and acts upon the riboswitch, which enables expression of geneIII. Additionally, the AP encodes for the chaperone HDJ-1, which is essential to receive the functional CYP1A2 enzyme (Fig. 1).
In a first step, we wanted to validate our AP. Therefore, we added theophylline with a concentration of 100 µM to our inoculated culture and performed two rounds of PREDCEL. Afterwards, we determined the phage titers by plaque assays. Our theophylline treated culture displayed approximately two times higher phage titers than the non-treated control culture. Using the same experimental conditions, but replacing the theophylline treatment by a 300 µM caffeine treatment, we verified the functionality of CYP1A2 and thus of our SP. If caffeine is added to the culture, CYP1A2 catalyzes the reaction from caffeine to theophylline. The resulting increase of the theophylline concentration further activates the riboswitch on the AP and phage propagation is stimulated (Fig. 2). For the evolution of proteins via PREDCEL the addition of a Mutagenesis Plasmid (MP) is essential. For our cytochrome engineering approach we have chosen MP4, which induces a medium mutation rate
badran2015development. After six iterations of our optimized PREDCEL workflow, we performed plaque assays and sequenced single plaques. The sequenced plaques showed five recurrent mutations demonstrating that we are able to induce mutations with our experimental setup and that we are able to evolve enzymes (Fig. 7).
Engineered highly active Beta-Lactamase mutant E102FThis part is an improvement of BBa_K1189009: Beta-Lactamase with His-Tag.The aim of our project was to generate protein functionality, in vivo through PACE and PREDCEL and in in vitro with help of our software suite AiGEM. To prove that our circuit works, we mutated ß-lactamases based on the scores of our software. Many different mutants were cloned and screened for their minimal inhibitory concentration (MIC). Visit our software validation site here. Typical methods to determine MICs are broth or agar dilutions. The principle is as simple as effective. The microorganism is cultured in liquid medium or on plates with different concentrations of the inhibiting agent
In our case, we want to evaluate differences in the activity of a well characterized protein. Consequently, such an dilution method is ideal. It is easy to setup, very robust and gives exactly the information that is needed for the characterization.
To start the assay, colonies were picked into LB medium with 100 µg/ml chloramphenicol, but without carbenicillin and were grown to the stationary phase for 20 h. Then, deep well plates with 500 µl LB broth per well were prepared. The medium was supplemented with 100 µg/ml chloramphenicol and for each mutant, eight wells with different carbenicillin concentrations, ranging from 10-1280 µg/ml were prepared. Each well was inoculated with 2 µl of the proculture. The new cultures were incubated another 8 h. Finally, the OD600 of each well was measured. By this measure we were able to assert the growth ability of each variant. A first screening revealed, that beside catalytically inactive variants, we also had many candidates that even could survive the highest carbenicillin concentrations (1280 µg/ml)(Fig.: 1). As a result, we decided to perform a second test for these candidates with higher antibiotic concentrations. The three highest carbenicillin concentrations of the first data set were included and the range was extended to a maximum concentration of 19.2 mg/ml. While several candidates turned out to have a weaker activity than the wildtype protein, five of them showed improved properties. The two best candidates could even grow at 19.2 mg/ml (Fig.: 2). The variants were contained one point mutation each, E102F and M67G. These most benefitial mutations, appear in many of our better candidates as well, which underlines there functionality. The outstanding candidate was the E102F mutant, which could even grow at 19.2 mg/ml without affection by the antibiotic. This results in a improvement of at least 100 % in comparison to the wildtype ß-lactamase. This remarkable gain of activity proves, that our software is not only able to recognize protein classes, but also to generate new function.
Parts CollectionWe present the first self-contained in vivo evolution toolbox comprising 26 standardized BioBricks. The collection includes several key aspects of our project:
The crucial step of to the cloning of PACE circuits is the generation of the accessory plasmid. These plasmids allow geneIII expression dependent on the evolving protein. The link between the fitness of the protein of interest and the expression of geneIII determines the effectiveness of the directed evolution and the presence of ProteinIII is essential for the production of new phage particles.
Therefore, we provide an accessory plasmid construction kit for phage library selection and propagation in E. coli based on conditional geneIII complementation. The geneIII-driving promotor, RBS and plasmid ori can thereby be fully customized.
If more than one variant of a circuit should be tested at a time, it is necessary to modify the activation region for geneIII and the additional gene that can be located on the AP with a minimum of effort. This is the another reason for a efficient cloning strategy.
To make AP cloning as simple as possible, we defined a new cloning standard that is specifically suited for the assembly of APs, we wrote a BBF RFC that describes our concept in detail (Fig. 1).
In Addition, an optogenetic selection stringency modulator comprising the light-dependent EL222 transcription factor and an engineered, hybrid Psp-EL222 promoter.
Furthermore, we provide parts for production of geneIII-deficient M13 phages carrying a custom gene-of-interest to be evolved.
On order to validate our system, we constructed the plasmids for our CRISPR/Cas9 subproject according to the RFC cloning standard. We could constuct several plasmids with help of this standard.
At the heart of our toolbox is an innovative workflow for enzyme engineering, paving the way towards bio-production of novel compounds. It comprises promiscuous enzyme candidates (CYP1A2 and Cytochrome c) and selection-mediating accessory parts such as our riboswitch for detection of an organosilicon (BBa_K2398555). Our simple PREDCEL protocol and accompanying online toolbox guide bring in vivo evolution to future iGEM teams and the broader synbio community.
Finally we created a set of parts, which were used in our different subprojects. These parts were transferred into pSB1C3 in a way that they are still compatible with our standard and submitted them to the registry: