The team documented our work over the weeks and compiled each week into concise summaries. See our progress over time below!
| Week | Lab Activity |
|---|---|
| Week 1: 5/15-5/19 | Researched medically relevant aptamers. We picked several proteins of interest that had previously created aptamers of 75 nucleotides or less on Aptagen. We picked Bovine Thrombin, Thyroxine, bFGF, VEGF, IL-32, and mCRP-1. IL-32, and mCRP-1 were also picked as a positive control for the creation of the other riboswitches because they have previously been created |
| Week 2: 5/22-5/26 | Read all relevant parts of the Addgene:Plasmids 101 textbook and made references for all the protocols needed for our project. Completed training in the lab consisting of practice with PCR, chemically competent cell prep, digestion, ligation, transformation, miniprep, and gel electrophoresis |
| Week 3: 5/29-6/2 | Started experimenting with the De Novo DNA Riboswitch calculator and got an understanding of how the calculator works. We then gathered all the information needed to use the riboswitch calculator on the 6 proteins of interest. |
| Week 4: 6/5-6/9 | We researched the effectiveness of Glucose Oxidase sequence from A.niger when translated in a cell free system. Our team also started to create riboswitches with a CDS of the Glucose Oxidase sequence from A.niger, thinking this would act as our reporter. Made CC cells in the lab |
| Week 5: 6/12-6/16 | Shifted our focus from glucose oxidase to Glucose Dehydrogenase in P. megaterium. We then created riboswitches with P. megaterium Glucose Dehydrogenase sequence, thinking this would act as the reporter as we compiled papers about FAD-dependent glucose dehydrogenase with our TXTL system. |
| Week 5: 6/12-6/16 | Shifted our focus from glucose oxidase to Glucose Dehydrogenase in P. megaterium. We then created riboswitches with P. megaterium Glucose Dehydrogenase sequence, thinking this would act as the reporter as we compiled papers about FAD-dependent glucose dehydrogenase with our TXTL system. |
| Week 5: 6/12-6/16 | Shifted our focus from glucose oxidase to Glucose Dehydrogenase in P. megaterium. We then created riboswitches with P. megaterium Glucose Dehydrogenase sequence, thinking this would act as the reporter as we compiled papers about FAD-dependent glucose dehydrogenase with our TXTL system. |
| Week 6: 6/19-6/23 | From the Riboswitch calculator, we selected five different riboswitches for each protein of interest based off of different OFF/ON translation rates, activation ratios, and binding constants. Ordered the G Blocks and oligos for a total of 30 different chosen riboswitches Transformed our pFTV1_mRFP1 plasmid into CC NEB5a cells Performed Miniprep on pFTV1-mRFP1, quantified, and saved a cryostock |
| Week 7: 6/26-6/30 | Received the majority (13/15) of ordered G-blocks and accompanying oligos/primers Tested effectiveness of various polymerases in the amplification of G-Blocks through analysis of concentration and purity after 3 PCRs with cleanups. Out of two different Q5 and one Taq polymerase, Q5 was found to be most effective. PCR of mCRP G-blocks 1-5 carried out with most effective Q5. Oligos annealed (bFGF, VEGF, IL-32y) Double digested pFTV1_mRFP1 vector with XbaI and SacI enzymes Vector purification quantified |
| Week 8: 7/3-7/7 | PCR and PCR cleanup with G-blocks of Thyroxine and Bovine Thrombin quantified Tested our ligation and transformation protocol to make one of our riboswitches (bFGF1) into PFTV1_mRFP. Sample was cryostocked and quantified through mini-prep. Since this procedure was successful, ligation and transformation protocol was carried out and quantified after mini-prep for bFGF 2-5 and VEGF 1-3 Digestion, gel, and PCR of pFTV1_mRFP vector mCRP 1-5 digested with XbaI+SacI, then cleaned up in gel and quantified Sequence confirmed pFTV1_bFGF(1-5)_mRFP |
| Week 9: 7/10-7/14 | More pFTV1_mRFP was digested and cleaned up for ligations and transformations VEGF (4-5), mCRP (1-5), and IL-32 (1-5) ligated into pFTV1_mRFP1 vector and transformed in NEB5alpha CC cells. For all cultures, sample cryostocked and minipreped for later use. Thyroxine (1-5) and Bovine Thrombin (1-5) PCR products digested and cleaned up for further ligations |
| Week 10: 7/17-7/21 | Carried out PCR, digest, gel, and PCR cleanup of BThrombin (1-5) in preparation of ligation More NEB5alpha CC cells were made from cryostock BThrombin (1-5), Thyroxine (1-5), bFGF (2,4,5) ligated into pFTV1_mRFP vector and transformed into NEB5alpha CC cells. Samples were then cryostocked and mini-prepped Sequence confirmed pFVT1_mCRP1_mRFP and found it had a dimer so it must be re-done In the Riboswitch calculator, codon optimized our new reported Tre-37 and ordered as a G-block Ordered T7 promoter as oligos |
| Week 11: 7/24-7/28 | Due to issues with lower than expected plasmid yields, we tested different incubation conditions, namely 37C 300RPM for 18-20hrs vs 30C 300RPM for ~24hrs. We found that longer incubation time was needed for a higher concentration. Due to Sanger Sequencing and digestions needing higher concentration of plasmids, culturing and miniprep of several clones was redone for mCRP1-5, Thyr1-5, BThrom1-5, and bFGF2,4,5. After mini-preps with longer incubation, concentration was still too low. |
| Week 12: 7/31-8/4 | To address the low yields of plasmids, more experimentation with pFTV1_mCRP1_mRFP only in LB and Terrific Broth (TB) media began with varying times, temperatures, reagents, and protocols. PCR and PCR cleanup of newly arrived Tre-37 G-block T7 Promoter oligo arrived and annealed 21x6mL LB+Cm35: 21 cultures made for multiple different riboswitch plasmids of: IL-32 y 1-5, BTHrom 1-5, mCRP 2-5, bFGF 2,4,5, 2xThyr 3-4 A mini-prep of each sample was performed and onces with desirable concentration were kept along with cryostocks. BThrom(1-2) , mCRP(1), bFGF(4-5), and IL32y (4) cultures were remade and mini-prep results were optimal. TXTL positive and negative control reactions set up and ran on Tecan overnight to test efficacy and protocol |
| Week 13: 8/7-8/11 | During our final week in the summer, we researched the correct protocol we should be using when characterizing our system. In the Tecan, we put our TXTL cell free system, our sequence confirmed plasmid with inserted riboswitch, and the ligand we are looking to detect through mRFP fluorescence. |
| Week 14: 8/14-8/18 | No activities took place in the lab this week. |
| Week 15: 8/21-8/25 | Reorganized plasmids and lab space as the new semester began Reassessed cloning and characterization plan |
| Week 16: 8/28-9/1 | Miniprepped and completed sequence verification for J23100/mRFP1 riboswitches that previously failed Began TECAN tests with J23100/mRFP1 Thyroxine riboswitches |
| Week 17: 9/4-9/8 | Began rapid cloning of T7/mRFP1 plasmids Performed minipreps, digestions, gel electrophoresis, cleanups, ligations, transformations, and platings for multiple rounds of 10 plasmids each. Troubleshooted TECAN and continued attempting thyroxine riboswitch tests |
| Week 18: (9/11-9/15) | Continued cloning of T7/mRFP1 plasmids Sent off new plasmids to be sequence confirmed TECAN experienced technical difficulties, so testing halted for the week |
| Week 19: (9/18-9/23) | Began cloning of T7/Tre37A plasmids for the no-riboswitch control, Thyr5, BThrom5, and mCRP3 Sent off T7/Tre37A plasmids for sequencing TECAN technical difficulties continued |
| Week 20: (9/25-9/29) | Troubleshooted poor sequencing results Recloned all failed T7/mRFP1 riboswitches besides VEGF and IL-32y since we did not have those ligands to test and had limited time Sent off plasmids for sequencing |
| Week 21: (10/2-10/6) | Minprepped from confirmed cryostocks to prepare all needed plasmids for fluorescence testing in TECAN Tested T7/mRFP bFGF and mCRP riboswitches in triplicate in TECAN |