Flappase Characterization
For the 2021 Giant Jamboree, we focused on contributing to the Flappase composite part (BBa_K3389003). The Flappase-10X His-tag composite part was an essential part of our project, and we chose to characterize the expression of the protein, as well as methods for lysis and purification of the part. The composite part included an inducible promoter a ribsome binding site, the gene for Rad 21 (aka Flappase) and a 10x His tag for purification.
We began with a series of small-scale expression and induction tests. To do this we induced with several concentrations of IPTG (0mM, 0.5 mM, and 1 mM) and harvested at different times. We found that maximal expression of the construct was achieved in BL21DE3 competent cells with 1mM IPTG and overnight incubation.
Along with the induction tests, we also conducted small-scale lysis tests to determine the optimal settings for lysis of the cells by sonication using a Qsonica Q-800R1 rotating sonicator with chiller. We tested sonication times of 1, 3, 5, 7 and 10 minutes. We chose sonication lysis for 3 minutes, at 60% amplitude, 20 seconds on, 10 seconds off. This sonication setting gave us the highest yield of protein from the induced cells.
We also conducted some initial experiments to work out a purification protocol using a nickel Immobilized Metal Affinity Chromatography (IMAC) column. The purification work done consisted of optimizing a protocol for optimal purification settings. The IMAC protocol went as follows: column equilibration (Buffer A: 300mM imidazole, 50 mM sodium phosphate), sample loading (1mL cell free extract), sample wash (wash 1, Buffer A with 20mM imidazole) and elution (isocratic, Buffer A with 500mM imidazole). Ideally, our protein of interest would be located in the elution sample. However, we suspect that Flappase is coming off of the column in the second wash stage, based on an SDS-PAGE analysis of all collected fractions that showed significant A280nm absorbance peaks in the chromatogram. These peaks of interest were analyzed and they showed a single, 42kD band. This corresponds to the expected molecular weight of the Flappase protein. Unfor tunately, due to time constraints we were not able to confirm this identity via Western Blot. The optimization lead to the realization that our imidazole concentration in the wash stage (20mM imidazole) is too high, and our protein of interest comes off the column prematurely.
Assessing Interest in Creation of an iGEM Hardware Registry
In our two years competing in iGEM, we have attempted to research various hardware items produced by other teams throughout the years of the competition. We intended on using the designs of other teams as a starting point for creating our own devices, much in the way teams use parts created by other teams to create biological circuits.
Through our efforts, we have come to find it unnecessarily difficult to locate, digest, and effectively utilize data that pertains to hardware devices. This difficulty is due to the lack of a central location housing device designs, forcing teams to search through individual wiki pages. The work is complicated by the lack of a search engine that can filter devices by name or type, and a lack of standardization when it comes to information about devices being distributed.
Given that iGEM has a Parts Registry for Biological Parts and Devices, we set out to investigate the usefulness of a similar registry for Hardware devices. Our goal was to gauge if the iGEM community has interest in such a registry, parameters of such a registry, and what types of standardized information should be included in this registry. We hope that such information might inspire the creation of a registry prototype on the iGEM server for use in upcoming competition cycles.
The survey that we designed initially contained 7 questions. We invited iGEM teams to participate in the survey through the 2021 iGEM Collaboration’s web page, directly emailing teams we have collaborated with on other projects, and through social media. As of 10/14/2021 15 teams logged answers to survey questions. We should note here that it is likely that more teams attempted to take our survey than were logged. The reason for this is that we accidentally misset conditions on one of the questions. This mistake led to teams that indicated that that they were not building a hardware device for the 2021 competition to be closed out of the survey. When we discovered the mistake, we removed this question to permit all teams to access the survey questions.
Survey Results
Question 1: How many years has your team competed in iGEM?
Question 2: Would a Hardware repository be useful to your team?
Question 3: Question 3: If you answered yes or maybe to the previous question, should entering data be a competition requirement or on a voluntary basis?
Question 4: Ranked choice - What should a Hardware Repository be named?
Question 5: Choose all that apply, and rank the importance - What fields should be included in a Hardware Repository?
Question 6: Ranked choice – Rank the importance of each field that might be included in a Hardware Repository.
Participants were also provided a free response section to include any additional comments. No responses were logged.
Conclusions regarding interest in a Hardware Repository
From the results of our survey, most of the teams that participated in the survey are interested in the development of a Hardware Repository and might make use of this tool. The majority of participants believe that participation in such a database should be voluntary. When it comes to the build of such a Repository, most teams favored calling it a Hardware or Instrumentation Registry. Teams generally agree that this should contain information on device description, schematic diagrams, links to corresponding wiki pages and attribution to the teams that designed the device, device features and characterization data, along with references. Teams generally ranked these items in the importance that is ordered.
Through conducting this survey, our team has concluded that a standardized repository of hardware devices is a tool that is favored by the iGEM community. We will be transmitting the results of this survey to iGEM HQ, with the hopes that they will begin development on this useful tool.