- We arranged an hour-long call with Dr. Stewart from the University of Utah to share our proposed project goals discuss caddisfly silk and gene properties and predict major obstacles. He also shared an unpublished article with information on the modular structure of the h-fibroin gene.
9 April (Team)
- After months of ideation and development we selected caddisfly silk as our Hopkins 2023 project!
April-May (Hardware)
- We have ideated designed and integrated new components and modifications to our Hopkins iGEM bioreactor which was first assembled in 2021. This came from substantial discussion and mentorship from Sreeni Eadara (Hopkins 2020-2022). So far we have taken the 3D printing software Marlin and modified it to work with a bioreactor allowing the user to modify parameters such as temperature and pH through a simple LCD interface--similar to how you can change extruder and bed temperatures with a 3D printer to modify output quality.
April-May (Hardware)
- Beyond the bioreactor our second substantial idea is for the design and assembly of a spinner kit. The goal is to process our raw silk/fibroin solution into a usable adhesive (spinner) that we can administer to a surface with controlled physical properties (applicator). We have proposed the design identified and ordered parts and are evaluating possible test materials.
April 16 (Dry Lab)
- We discussed ideas for modeling our protein and planned a promotional video to show to the JHU Student Initiatives Fund to present our teamÆs activity to JHU engineering faculty.
April 30 (Dry Lab)
- We submitted our promotional video for the Student Initiatives Fund.
8 May (Wet Lab)
- We received h-fibroin and l-fibroin sequences from Dr. Russell Stewart.
27 May (Wet Lab)
- Russell Stewart sent us a paper which contained a script to visualize the locations of serine-rich repeats in the sequence. However we were unable to get the script to work properly.
May-Jun (Wet Lab)
- We discovered that the heavy chain sequence was too long for a feasible plasmid. Research into shortening our sequences by truncating highly repetitive regions.
1-15 Jun (Hardware)
- Conceptualization and Design: We finalized the design for the portable bioreactor focusing on simplicity affordability and integration possibilities for sensors like pH and optical density. We successfully integrated the temperature sensor into the bioreactor and we have added CAD designs and have 3D-printed new parts for the system.
Jun-Jul (Hardware)
- We contacted Dr. Yayuan Liu @ the Liu group (at JHU) to help us with all electrospinner inquiries. She allowed us to test with her electrospinner and learn the intricacies of how it works. For the glue gun we are working out the details of including a microfluidic device. We have developed a model to determine the force and shear to justify using microfluidics over conventional pressurizing methods and are looking for ways to improve the parameters/outputs.
3 May - 19 July (HP)
- We contacted Drs. Paul Frandsen and Gabi Jijon (introduced to us by Dr. Stewart) to find out more about available gene sequences for L- and H- fibroin and obtaining silk samples.
27-29 May (Wet Lab)
- We requested caddisfly samples for rtPCR of our target sequences and we researched BACs and other methods to transform larger plasmids into a chassis. From this we formulated our first experimental plan.
28 May (Dry Lab)
- We modified a script provided by Dr. Stewart to visualize repeats in the H-fibroin sequence.
30 May (Dry Lab)
- Submitted a request to access NovaFold AI for more powerful protein visualization.
30 May (HP)
- We shared our new experimental plan for reduction of repeats with Frandsen Stewart and Jijon for discussion and feedback.
31 May (HP)
- Meetings and feedback from Dr. Stewart on the experimental plan. He provided key information on the known locations of repeats in several species-specific h-fibroins and also suggested that we investigate the requirement of disulfide bond formation and phosphorylation in our E. coli.
1-10 Jul (Hardware)
- Material Procurement: We ordered a mason jar 3D printing materials pH sensor optical density sensor and other necessary components for the bioreactor project.
6 Jun (Dry Lab)
- We developed additional scripts and used repeat finding and local alignment tools to identify repeat motifs. We indexed occurrences of these repeat motifs as candidates for removal from the coding sequence.
9 Jun (Dry Lab)
- Met with Carl-Erik Tornqvist from DNASTAR about visualizing our H-fibroin using tools such as NovaFoldAI and I-Tasser. However we decided not to go forward because there was no guarantee the software would work on our model.
10 Jun (Wet Lab)
- Discussion of the post-translation modification problems. We adjusted the wet lab plan switching our species to G. pellucidus and using a synthetic truncated sequence in lieu of rtPCR.
24 Jun (Wet Lab)
- We completed manual annotations on the entire heavy chain of G. pellucidus and we designed a truncated control h-fibroin sequence.
3 July (Dry Lab)
- We tested the script from Calgary toward reducing truncated sequences though this was not our final approach.
3 July (Wet Lab)
- We finalized our protocol for disulfide bonding in T7 Shuffle Express competent E. coli. We also designed two longer truncated h-fibroins informed by gene annotations. With viable truncated sequences we eliminated the requirement for BACs.
12 July (Dry Lab)
- We did a preliminary red and blue visualization of the GC content of our sequences using a MATLAB script we wrote. This further guided our efforts to truncate our h-fibroin sequences since we could see the frequency of the GC content.
16 July (Dry Lab)
- We further modified the MATLAB script to output both frequency and percentages of the GC content over a 125 base pair sliding frame. This helped us understand the complexity of trying to reduce the GC content.
1-15 Aug (Hardware)
- 3D Printing and Sensor Calibration: We 3D printed the housing for electronics in the bioreactor. We calibrated the pH and optical density sensors for accurate readings.
21 Aug (Wet Lab)
- We conducted an inventory of all the supplies in our lab to determine what we needed to order. Our ProMega plasmid miniprep kit had enough reagents to miniprep eight samples. We found that we were short on outgrowth medium and out of LB.
23 Aug (Wet Lab)
- We resuspended and transformed pJUMP29-1A pJUMP29-1B pJUMP29-1C pJUMP29-1D using kanamycin plates for selection.
24 Aug (Wet Lab)
- After the transformed pJUMP bacteria incubated on the plates for 24 hours we took our plates out of the incubator wrapped in parafilm and put in 4C chilled storage.
31 Aug (Wet Lab)
- We inoculated liquid LB with the transformed pJUMP29 colonies to grow liquid culture.
1-10 Sep (Hardware)
- Chassis Assembly and Sensor Integration: Assemble the bioreactor components into the mason jar. Integrate pH and optical density sensors securely into the system.
1 Sep (Wet Lab)
- We miniprepped the four pJUMP29 parts that we inoculated one day before. We stored the purified DNA in the -20C freezer.
27 Sep (Wet Lab)
- The following supplies arrived at the lab: spectinomycin plates miniprep kit SOC outgrowth medium LB and chloramphenicol plates.We transformed the pJUMP49-2A part along with mCherry ribosome binding site and terminator using chloramphenicol or spectinomycin for selection. We then took the colonies and inoculated media to grow liquid culture.
29 Sep (Wet Lab)
- We miniprepped the four parts that we transformed two days previously and measured concentrations (ng/uL) A280/260 and A260/230 ratios (to check sample quality/purity) using the nanodrop machine. We stored the purified DNA in the -20C freezer.
30 Sep (Wet Lab)
- We resuspended the promoter and transformed the bacteria using ampicillin plates for the selection.
1-15 Oct (Hardware)
- Electronics Integration: We integrated electronic components into the 3D printed housing including microcontrollers for sensor readings and control systems (pH buffer OD600 temperature oxygenation readings).
1 Oct (Wet Lab)
- We inoculated LB media with the promoter colonies.
2 Oct (Wet Lab)
- We miniprepped the promoter plasmid
3 Oct (Wet Lab)
- We assembled the level 1 vectors using the golden braid protocol.
4 Oct (Wet Lab)
- We transformed the level 1 vectors into bacteria
5 Oct (Wet Lab)
- We inoculated LB media with colonies from the four level 1 vectors and noticed the colonies were green but if the assemblies worked the cells wouldnÆt express GFP. We decided to do a diagnostic restriction digest to check if our BsaI enzyme works.
6 Oct (Wet Lab)
- We miniprepped the four level 1 vectors that we transformed two days previously and measured concentrations (ng/uL) A280/260 and A260/230 ratios (to check sample quality/purity) using the nanodrop machine. We sent 12uL to Plasmidsaurus for whole plasmid sequencing. We stored the rest of purified DNA in the -20C freezer. We set up a diagnostic restriction digest to check if our BsaI enzyme works.
8 Oct (Wet Lab)
- We ran the products of restriction digest with BsaI on a gel and compared the resulting bands with expected bands if BsaI worked properly. We received the results of the whole plasmid sequencing and confirmed that our samples from the level 1 assembly contained GFP. We decided to order a new vial of BsaI enzyme to repeat the level 1 assemblies.