Wet Lab
Overview
Cornell iGEM is contributing various basic parts from the perchlorate reducing operon found in D. Aromatica. pcrA, pcrB, pcrC, and pcrD are the four subunits of perchlorate reductase . In addition, the team characterized the effect of a lipoprotein knockout to increase expression of periplasmic proteins. Finally, to integrate seamlessly with our product development team's ARC bioreactor, we characterized the effect of curli genes to increase biofilm formation of E. coli .
Parts:
Name | Type | Description | Designer | Length(bp) |
---|---|---|---|---|
BBa\_25JIB2TT | Basic | pcrA from D. aromatica | George Zeng | 2694 |
BBa\_25SAHPIE | Composite |
pcrA from D. aromatica, codon optimized for E. Coli . Contains native E. coli TAT tag for transport to the periplasm and His-tags for purification.
Components: Shine-Dalgarno region E. coli TorA signal peptide (Part:BBa\_K3114005) His-tag |
George Zeng | 2879 |
BBa\_25TE3V5C | Composite |
pcrBCD from D. aromatica, codon optimized for E. Coli . Contains native E. coli TAT tag for transport to the periplasm and His-tags for purification.
Components: Shine-Dalgarno region E. coli TorA signal peptide (Part:BBa\_K3114005) His-tag |
George Zeng,
Anousha Shadid, Aaryaa Chiney, Antone |
2952 |
Composite Parts:
Lipoprotein Knockout
E. coli on its own is often ineffective in expressing large proteins due to the lpp gene, which encodes Braun's lipoprotein (lpp). This protein stiffens the cell membrane of E. coli by covalently cross linking the outer layer of the membrane with the peptidoglycan layer (src). By knocking out this protein, the membrane becomes more permeable and large proteins can be expressed more freely. Lpp knockouts and mutants have previously been used to enhance the excretion of small molecules such as in organophosphate hydrolysis and L-carnitine synthesis (src). Using an lpp knockout strain is crucial to our project as we hope to engineer E. coli to reduce perchlorate by expressing the perchlorate reductase (Pcr) gene block, which is fairly large with many enzymes (src). In addition, in order for the catalytic domain of Pcr to be functional, it must be localized to the periplasm, which we hope to ensure using an lpp knockout.
Our use of the lpp knockout is a novel contribution to the literature. While many lpp knockout applications focus on the release of peptides into surrounding media (src), PRoSPER instead leverages the increased periplasmic space for greater expression of membrane-linked enzymes. Through the expression of additional auxiliary membrane-embedded linkers such as quinol dehydrogenase tetraheme cytochrome (QDH) and diheme c-type cytochrome (DHC), we ensure that perchlorate reductase is still anchored to the cell membrane and accessible to the electron-carrying quinone pool necessary for reduction.
Additionally, lpp can be beneficial to the co-culture of these bacteria. Synechococcus PCC. 7002 sequesters Cl-, takes in CO2, and outputs organic carbon, while the E. coli takes in organic carbon and perchlorates, then outputs water, Cl-, and CO2. This closed-loop system relies on the successful secretion and uptake of metabolites by E. coli , which would be further enhanced by the lpp knockout. This application of an lpp knockout to improve a bacterial co-culture is also a novel contribution to iGEM and broader research.

New Composite Part - TAT System
Important genes for perchlorate reduction, such as Cld, PcrA, PcrB, and PcrC, are transported to the periplasm using twin arginine translocation (TAT) signal peptides and the bacteria's TAT translocase, which transports folded proteins across the cell membrane. Exeter 2016 verified the efficacy of this TAT system by attaching GFP to target signaling peptides and showing that these fusion peptides had reached the periplasm. We will build upon this knowledge by applying the TAT system to subunits and enzymes central to the reduction of perchlorates in martian soil. In particular, with Pcr, the TAT system simultaneously allows its molybdenum cofactor subunit to be cytoplasmically integrated during folding, while other subunits are localized to the periplasm.
Culturing Guide of Synechococcus PCC. 7002
This particular strain of Synechococcus PCC. 7002 was chosen for its short doubling time and demonstrated ability in past literature to be used in desalination processes. We obtained our seed stock through a generous sponsorship from Cultivarium and ATCC. We initially began culturing our Synechococcus in 25 mL liquid cultures under 24 hour LED plant grow lights, but found that they could not be sufficiently aerated for the necessary gas exchange. They grew slowly over two weeks before becoming increasingly yellow in color until growth appeared to stop.
We then decided to follow ATCC's culturing recommendations of agar slant and plated cultures. Following slant preparation protocols from Caprette (2016), we prepared several cultures within 15 mL screwcap tubes. We found that a 1% agar gel was best for slants, while 1.25% was best for plate preparation. One 25 mL liquid culture was spun down to concentrate cells at the bottom, forming a loose pellet. The concentrated stock was used to inoculate the new cultures.
Despite the low growth, we began a wet run with the ARC-P bioreactor. We inoculated 700 mL of media with our liquid cultures. All conditions were optimized for Synechococcus growth, but we weren't seeing any growth after a couple days.
As mentioned in our Engineering Success page, we then found that B12 is crucial for Synechococcus growth. Our final and most effective culturing method has been 10 mL liquid cultures in 15 mL tubes with the opening covered by a permeable material such as a Kimwipe. The tubes are cultured at a slant for optimal light exposure and gas exchange. In future experiments, we hope to use these to inoculate a new 700 mL culture in the ARC-P bioreactor.

Characterization of Synechococcus PCC. 7002 and Co-culture with E. coli
An important aim of PRoSPER is to characterize the growth of Synechococcus PCC. 7002, and the metabolite linking cycle between Synechococcus PCC. 7002 and the transformed E. coli . Creating a co-culture between these two organisms allows us to perform toxin cycling to reduce perchlorate levels and salinity in Martian regolith, as well as nutrient cycling to make the co-culture more self-sufficient. In this closed-loop system, E. coli reduces perchlorate (ClO4-) to chloride (Cl-) using organic carbon as an energy source. The cyanobacteria Synechococcus PCC. 7002 takes up chloride with atmospheric CO2 to produce organic carbon through photosynthesis, which can then support the growth of E. coli . For the first step to successfully characterize this co-culture, we modeled the growth of E. coli independently to observe how perchlorate concentration affects its growth (Figure 2). Future experiments will be conducted in conjunction with Product Development to assess and optimize the metabolite linking system for both organisms, taking into account their individual growth requirements. First, Synechococcus PCC. 7002 will be cultured and monitored in BG-11 media, which is suitable for cyanobacteria. Later experiments will include engineering E. coli to be resilient in high salinity and high perchlorate conditions, monitoring growth in the co-culture using absorbance and chlorophyll fluorescence, and measuring the overall perchlorate reduction rate.

Product Development
Adaptive Reaction Chamber Family
The Product Development team designed and built the Adaptive Reaction Chamber (ARC) family for the PRoSPER project. Its original goal was to meet PRoSPER's deployment requirements for culturing two model organisms and conducting soil remediation. After multiple design iterations, we developed a fully modular, laboratory-scale microbial cultivation platform for the synthetic biology community. By assembling different modules, ARC can rapidly adapt to various microbial cultivation needs. Transforming from a CSTR to a photobioreactor takes only 30 seconds. This design serves as a comprehensive bioreactor solution for Cornell iGEM and synthetic biology community worldwide.

The key to ARC's quick modular assembly lies in its interchangeable modules. Using MagPin, a 5-pin magnetic connector, we link the electronics of peripheral modules to the reactor's core circuits, enabling quick module replacement. For detailed module descriptions and demonstration videos, please visit Hardware page.



The following is the general user manual including operation guide and assembly guide for the ARC bioreactor family. The user guide covers how to initialize the reactor, perform an autoclave, configure modules according to experimental needs, and main structure assemble instructions.
We also conducted subsystem testings to all the modules, sensors, and the soil flowthrough system. The followings are the protocols:
One of the major advantages of modular design is the ability to continuously expand with new modules. In conversation with many of our stakeholders, such as Dr. Kate Scow, we considered various steps of bioreactor design. We hope that, with the support of the synthetic biology community, ARC will continue to grow through future module updates. This is also why we are sharing all ARC 3D files.
The following are the STL files for ARC and Modules:
Controls: Left click + drag to rotate | Scroll to zoom | Right click + drag to pan
We recommend printing the modules vertically for the best results. For detailed assembly instructions, please refer to the Engineering Success section or the user manual.
Our vision is that any iGEM or lab team can quickly get started with the ARC bioreactor using the above documents. With just a 3D printer and some basic electronics knowledge, anyone can build their own ARC.
Policy and Practices
Space Policy Handbook
Together, our team created a space and ethics policy handbook. We were originally inspired for this handbook by various interviews and our policy research. As a team, we found that there was no universal law or policy about space and space colonization. Instead, various countries had their own policies and views about how space exploration should be executed. This motivated us to create a handbook that includes various laws and policies from countries around the world in order to streamline access to this kind of information. We also proposed laws and policies we thought could be helpful in the ongoing debates and discussions surrounding this kind of legislation. Our suggestions were inspired by our own personal research, conversations we had with people during outreach events, and stakeholder input during interviews.
We hope that this handbook can be utilized by our teams that compete in the space village in the future. Currently, there is not a lot of research and time put into space law and legislation and we hope that this is a starting point to open up conversations with other iGEM teams about how we can tackle the policy ethics of space travel.
Podcast
This season, our team premiered our podcast on Spotify. We were inspired to start this podcast to bring educational information to people in a more digestible format. A lot of times, people do not have time or interest in reading about science development, so we thought that this could be an interesting new medium to help relay important information to the public. On our podcast, we interviewed various team members about different topics like our brainstorming process or wet lab protocols. We also made educational episodes about PRoSPER and how our research is important to the world. Not only this, but we also interviewed other iGEM teams from different universities, like the University of Michigan and the University of Maryland. This helped us bring in different perspectives to our podcast and help our listeners learn from a variety of different people.
We hope that we can continue to use this podcast to educate not only the public but also release information into the iGEM community. A lot of times, our podcast episodes focused on things like how to get interviews or how to build a digestible wiki. We found that these conversations, especially those with other teams, were very helpful for iGEM teams overall, which was also a big motivation for us to post these episodes publicly. These episodes can be used by university students around the world who are looking to start an iGEM team but just are not sure where to start.
Social Media
Another important aspect of our education and outreach efforts was our social media. We had two main forms of social media this season: both Instagram and LinkedIn. Our Instagram had many educational posts about PRoSPER and synthetic biology, along with lighthearted posts about our team's culture. Our LinkedIn took a more serious approach, posting our educational materials along with infographics.
On both of our social media platforms, we were able to build a community with other iGEM teams. Through this we were able to connect with other teams and answer questions and collaboration requests that other teams sent our way. Being active on social media allowed us to interact and contribute to the community by sharing helpful posts, like ones that outline our methodologies. Our social media efforts are ongoing, and we post on our social media throughout the year. We hope to continue to post both educational and fun content in the upcoming seasons to strengthen and foster a sense of community within iGEM.