During the progress of our project, we communicated with researchers of various research focus and improved our design based on their insights. 1) We interviewed the potential users of our system, and learned that mutation rate and labor cost are their major concerns. We then addressed both issues in our system design. 2) We discussed with researchers working on prokaryotes and received valuable feedback. Prof. Lin warned us of the false-positive caused by Cre, which inspired us to attach a degradation tag to it. Prof. Alper confirmed the necessity of capsid protein for efficient reverse transcription. 3) Modeling benefited as well. With help from Prof. Huang, we established the design of dividing our system into three sub-models. 4) In addition, we categorized and organized the HP approaches of previous projects and released a guidebook for collaborators and future teams.

Interaction is our key focus. We divided our audience into three categories: kids, students, and the general public. 1) For kids, we introduced the concept and function of DNA through workshop. In the process, we established a long-term collaboration with Fudan Jiuqian Volunteer Association. 2) For students, to inspire them to pursue biological research, we held three lectures on synthetic biology, and one workshop to share lab experience. We also designed a boardgame to enlighten them with the mechanisms and significance of continuous evolution. 3) For the general public, we aimed to popularize biology - we distributed pamphlets with examples of how synthetic biology advancements have changed our lives, and published a video on PCR procedures to convey that the important discoveries were made from "down-to-earth" experiments. 4) In addition, we released a HP guidebook through cataloguing previous projects, which we distributed to collaborators and hope to guide future teams.

In our modeling, we successfully simulated the function of our mutagenesis system, and contributed to improve our experimental setup. We divided our system into 3 sub-models: induced expression model, reverse transcription model and Cre recombination model. We utilized deterministic and stochastic techniques with parameters derived from our experiments or published papers. Our modeling established theoretical basis for our experiments: 1) The determination of simultaneous or separate expression of reverse transcriptase and Cre is guided by models revealing their different working concentration. 2) We estimated the optimal expression level and induction time needed to achieve maximal recombination efficiency. 3) We modeled the effect of Cre degradation on recombination. 4) We demonstrated that mutations accumulate accompanying E. coli growth. Modeling acted as a shortcut of answering questions concerning experimental setup and revealed new insights into our system. Thus, we believe that our modeling work is very competitive for the best modeling prize.

We focused our measurement on characterizing the fluorescence recovery of EGFP from its nonsense mutation in the following 4 ways: 1) Green fluorescence could be seen on the plate under UV light through naked eyes and recorded by a cellphone camera. Liquid culture could be placed in a culture dish and fluorescence is easily detectable under fluorescent microscopy. 2) We designed PCR primers to only amplify the recovered EGFP sequence but not the mutated version. The amplified band could be easily visualized after electrophoresis. 3) Fluorescence level was quantified through microplate reader according to fluorescein solutions and silicon beads, both standard samples are from the distributed measurement kit. 4) We ran PAGE gel of IPTG induced bacterial lysates. The mutated version produced a truncated protein at 17.8 kD, while the recovered EGFP is 26.9 kD. We used multiple methods to ensure that EGFP is truly recovered from its nonsense mutation.

Our software simplifies the primer design process for target-specific mutagenesis via reverse transcriptase (RT). We called it tRNA primer designer. Studies have shown that tRNA functions as the primer for in vivo reverse transcription initiation: the 5' end of the tRNA interacts with RT, and the 3' end matches with the mRNA encoding the target. The software consists of 4 parts: reverse transcriptase selection, target sequence input, designed-tRNA visualization, and primer output. Although we only test MMLV-RT experimentally, the software can adjust its designing method based on the properties of well-studied RT from 3 species, MMLV, HIV-1 and RSV. Users could design their tRNA primers even for eukaryotic experiments. In addition, we calculate and output the tRNA acceptor stem annealing temperature, as this might be used as an indicator for likelihood to success.

To track bacteria growth on the plate and observe the fluorescence recovery from nonsense mutation due to continuous mutagenesis, we devised this hardware - the Fluorescence Tracker. It provides continuous, hands-off recording of the growth of plate colonies as well as fluorescent protein expression. For users of our mutagenesis system, with the help of our hardware, they could plate, and then monitor all plates together to increase the likelihood of spotting bacteria colonies with recovered fluorescence at the earliest time point. After discussions with our PI, we improved our hardware by adding remote access through TeamViewer, which allows visualizing the dynamic changes on smartphones. Although the current hardware is only suitable for monitoring fluorescence recovery, it could be easily modified to monitor bacteria colonies growing out of any antibiotic plate. Our hardware allows us to come to lab knowing that a plate with desired colonies is waiting for us.

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We provide a toolbox for in vivo site-specific continuous mutagenesis. 1) We attached Cre with different degradation tags, which could be used according to user's interest to achieve optimal recombination efficiency. 2) We placed MMLV-RT under different IPTG-inducible promoters, which provides a range of different steady-state expression levels for various experimental purposes. 3) We included additional regulatory sequences (e.g. native primer binding site and poly-purine tract) required for the initiation and completion of reverse transcription. 4) To eliminate self-excision of Cre and promote recombination efficiency, we included a set of incompatible loxP sites. 5) We provide testing plasmids for system verification and optimization. In summary, our part collection provides a complete set for assembly, test, and optimization of continuous mutagenesis in different prokaryotic hosts.