June 2023
In June of 2023, we started our CerviCare research journey. We completed our in-person wet lab work training for students who would conduct research at the University of Maryland’s Institute of Bioscience and Biotechnology Research (IBBR). The chosen biosensor for cervical precancer at this time were Volatile Organic Compounds (VOCs). The wet lab committee also explored the option of genetically modifying a bacterial host to incorporate a designed biosensor genetic circuit. The design committee familiarized themselves with Adobe Premiere Pro and established both a mockup the mid-atlantic meetup poster and a Wix site. The finance committee began applying to grants.
July 2023
5 July 2023 - 15 July 2023
Upon meeting with our advisors, Dr. Kahn and Dr. Eisenstein, we discussed the 2019 iGEM Sastra Team’s project, from which we drew inspiration for our project. We explored the future idea of implementing multiplexing into our project, as well as migrating from the use of VOCs to micro-RNAs (miRNAs). Testing with multiple miRNAs that produce various color outputs would avoid false positives and negatives. The process of establishing mock patient samples was introduced. Contrary to what was found in literature review papers at this time, an in vitro, cell-free system to test our toehold switches. The target miRNA detection response would be an increase in chromoprotein production for a visual output. This all pertains to the project goal of achieving a less invasive and expensive screening device for point-of-care use. Research into cervical precancer screening access began; the project then divided into two specific development phases: the toehold switch and the synthetic RNA device. Following the development of the RNA device, the plan entails designing a toehold switch, combining it with a reporter gene using Gibson assembly, and integrating these components into a functional genetic construct. This construct will then be coupled with a cell-free expression kit to enable reporter gene expression upon switch activation. To facilitate storage and transportation, the expression system will be lyophilized onto filter paper. Testing will include sensitivity, specificity, and detection limit assessments, using concentration studies and synthetic urine samples to evaluate the biosensor's response to target compounds or miRNAs. Additionally, a device will be designed to house the paper sensor, facilitating sample application, signal readout, and potentially incorporating additional features like control elements or data analysis capabilities.
18 July 2023 - 19 July 2023
Within these two days, the wet lab worked on introducing positive and negative control miRNAs to our research. There was also discussion on predicting the twister ribozyme folding in miRNA and aptamers with varying gates.
23 July 2023 - 24 July 2023
We received a $7,000 grant from Dr. Ian White from the University of Maryland Bioengineering Department! With the Mid Atlantic Meet up approaching, we started working on our presentation. We also had a meeting with our advisor, Dr. Khan, where we discussed how our system is going to work. The RNA structure stability, RNA sequences for chromoproteins, a possible inducible promoter, ways to produce variability and randomness, discussed on the decision of one plasmid or two plasmids to make miRNA were all parts of our project we focused on. We also made progress on the Gibson Assembly. Using the Gibson method to clone out small fragments and Gibson each into separate targets.
25 July 2023
On this day, Wet Lab worked on our Toehold. Our approach involved utilizing RNA structure site analysis and NUPACK for design. We aligned with the team's use of the same RBS and linker sequences. Images showed the AUG codon as a constant element, accommodating variable target sequences. We explored multiple tracks, employing three different linker sequences for each miRNA, initially with the GFP gene. Complexes with miRNAs had thermodynamic advantages but also introduced secondary structures affecting the RBS. We used LacZ as a detector with the Blue-White Assay, acting as a placeholder for linker sequence generation. The positioning of the linker before the reporter protein's coding region was significant. We drew inspiration from a Green paper for designing toehold switches for miRNAs and introduced a long A sequence in the miRNA 155 5p linker. Regarding multiplexing, we considered using AND gates and acknowledged that getting individual functions to work effectively before attempting multiplexing was essential, either in different wells or within a single cell
28 July 2023 - 30 July 2023
The Grant Project Abstract was completed, along with the Dojo Grant submission. Modeling worked on coding in R and Sequel for practice. Our wet lab now has all of the supplies needed for the toehold, the negative control made along with sequences and bifold designs. Outreach has begun coordinating date/time for a meeting with Jenna Mueller, and preparation for the meeting with Will Stone on August 2nd.
August 2023
1 August 2023
Finalizing what will be done in the lab once all materials have been received from IDT. Plan was made to grow a bacteria culture that will eventually express toehold switch and possibly ribozyme. Developed a plan to ensure our design is concentrating our data, checking if the screening device we create only reacts to target miRNAs, how much trigger miRNA will produce how much of a signal, and how much the sensor concentration affects the signal's strength. Lastly, to be sure to ensure that the lacZ gene produces a color that is easy to identify. We also discussed possible ways to validate our sensor activity, such as seeing if our switches/ribozymes will cause a change in protein expression in the presence of their targets and checking the orthogonality of our sensors, to make sure non-trigger RNAs do not cause expression.
5 September 2023
Today was Wet lab orientation at Kahn lab where Dr. Kahn reviewed lab safety information and showed us around all of the lab spaces. Modifications were made to the gBlocks canceled by IDT.
6 September 2023
Graham, Sarah, Rebecca, and Krista went into the Kahn lab to prep plates, buffers, and other necessary materials for the lab to begin. The modified gBlocks were ordered from IDT.
7 September 2023
The Ribozyme Report was started, and our committees began to work on the Wiki. With the beginning of a new school year, there were new members recruited to iGEM, so we went over how to pitch iGEM to potential new members.
11 September 2023
Today we had a meeting where we introduced new members to iGEM. On our design committee, the final cervicare promo video script was made.
In the lab, we began streaking E. coli cells and measured plasmid concentrations. Also, Dr. Khan bought the NEB Hifi kit from a loading dock in the Biopsych building.
13 September 2023
In the lab, we reviewed plasmid spectra sheets from plasmids and developed procedures for resuspension of gBlocks per IDT instructions, and doing digestions and run gels.
14 September 2023
On our design team, the promo video was completed and submitted.
17 September 2023
In lab, we redid Digestions and gel electrophoresis on controls.
18 September 2023
On this day as a general body, we shared project updates, gave wiki recommendations based on iGEM official website, and then we had time for committees to gather in groups to work on their wiki sections.
In the lab, we did colony screening and verification.
19 September 2023
IDT was not able to synthesize 1-miR-199-5p.
20 September 2023
In the lab we worked on Gel DNA Extractions using Qiagen protocol to purify and isolate the vector background.
25 September 2023
During our general body meeting, went over committee wiki roles and the last three months of the iGEM cycle calendar, and created the Digital lab notebook.
27 September 2023
In the lab we resuspended gBlocks at 50 nm.
October 2023
2 October 2023
Gibson Assembly for Toehold Switch insertion and concentration calculations were done in the lab.
3 October 2023
Outreach meeting with Sharmistha Mohapatra.
9 October 2023
Gave time for committees to gather in groups to work on their wiki sections.
Had an in-office day to re-analyze toehold sequences in the lab.
10 October 2023
Outreach meeting with Bryan Brensinger for follow-up and final presentation clarifications and advice.