iGEM 2023 — Team pennstate

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{% block title %}Notebook{% endblock %}
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# Notebook

## Notebook

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 |

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# Engineering

## Engineering

Combining biology with engineering is what makes synthetic biology so distinct. Applying an engineering approach to solving problems in biological systems allows synthetic biologists to revolutionize life itself—quite literally!

The Desing-Build-Test-Learn cycle is one of the key principles of engineering. Read on to see how the Penn State iGEM Team employed this as a strategy to engineer our diagnostic.

![](https://static.igem.wiki/teams/4950/wiki/eng1.png)

Figure 1: The Design-Build-Test-Learn (DBTL) Cycle visualized as a plasmid map.

## Research

Our team set out to create a diagnostic system for medically relevant proteins of interest in blood. To create the this, we wanted to build on previous work done in our lab under Dr. Salis. The backbone of our project originated from work done by Grace Vezeau, a Salis Lab graduate. Her paper validated the De Novo DNA Riboswitch Calculator for predicting effective riboswitch sequences that either express or repress a reporter, as shown through fluorescent characterization{1}.

The other significant piece to our concept stemmed from Keith Pardee’s work with genetic circuits in cell-free systems. Pardee explores how to make low cost diagnostics more practical through the use of glucometers. The experiment demonstrated the effectiveness of the trehalase enzyme in a glucose-based reporter system. Trehalase was shown to cleave trehaslose and produce glucose in a cell-free system, which was then measured with a glucometer[2]. This work was crucial to the structure of our idea of connecting our modular riboswitches with trehalase expression.

## Design (Cycle 1)

Based on the research we conducted, our team wanted to create a diagnostic system not through the measurement of mRFP fluorescent protein but through the detection of glucose through a glucometer in a cell-free system. We projected that our two biggests constraints would be the amount of myTXTL cell-free expression kits we had and the time we had to complete all the tests.

With constraints in mind, we designed our final cell-free system to include the following:

1. The biomarker (protein or small molecule) of interest
2. Modified pFTV1 plasmid
3. myTXTL transcriptional/translational factors
4. Trehalose

In theory, the protein of interest would bind to the aptamer region on the modified PFTV1 plasmid, which would create a conformational change, exposing the ribosome binding site (RBS). Exposing the RBS would allow the synthesis of trehalase. Finally, the produced trehalase would cleave the trehalose in the system and produce glucose molecules. The glucose would then be detectable through a instant glucometer reading.

However, we knew that to get to our final cell-free system design, we would have to create a multistep process to avoid changing more than one variable at a time. We decided for the first round of testing that we would measure the expression of mRFP1 fluorescent protein in a cell-free system with the same protocol followed in Grace Vezeau’s paper.

After creating a framework, our team needed to select the proteins of interest we would be testing. Our team utilized this previous research to set the parameters we would look for when picking our own proteins to detect. We researched proteins with previously tested RNA aptamers, optimally 70 base pairs or less, found in the blood and related to medical conditions. Using that criteria, our team decided to design riboswitches for six different proteins: basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bovine thrombin, thyroxine and two previously used proteins in Vezeau’s paper, Interleukin-32γ (IL-32γ), and monomeric C-reactive protein (mCRP). The selection of the last two proteins would act as a control group because no variables were changed from her paper.

## Build (Cycle 1)

We utilized the De Novo DNA Riboswitch Calculator to build our riboswitches. This calculator is a physics based model that uses statistical thermodynamics to predict DNA sequences and their functionality as a riboswitch that activates gene expression. From the list of DNA sequences given by the De Novo Riboswitch Calculator for each protein of interest our team picked five different sequences with varying activation ratios. Once we picked our riboswitches, we ordered all 30 off of IDT as oligonucleotides and PCR assemblies, depending on their size. Our next step was to insert them into our pFTV1 vector and clone them in E. coli.

Step 1:
Once we received our oligos and gBlocks, we performed double digestions with XbaI and SacI and ligated the various riboswitches into our vector.

![](https://static.igem.wiki/teams/4950/wiki/eng2.jpg)

Figure 2: pFTV1 plasmid template with J23100 promoter, riboswitch insert, and mRFP1 gene.

## Test (Cycle 1)

In the first round of testing our cell-free system included:

1. Our plasmid, pFTV1\_mRFP with our inserted riboswitches
2. My TXTL translational/transcriptional factors
3. Protein of interest

During this first test, the fluorescence of mRFP was analyzed in the TECAN Spark plate reader.

## Learn (Cycle 1)

In this round of testing, we learned that experiments will rarely go expected to plan. Our plasmids did not produce strong fluorescent signals, and we were unsure as to why. These results forced us to go back to our original design and pinpoint a weak spot.

Additionally, due to limited supply of myTXTL reactions, we did not run the samples in triplicate. With the given timeline and resources available, our team needed to reduce our remaining testing and focus on the most promising riboswitches, which were for bFGF (designs 2 and 3) and mCRP (designs 1-5).

## Design (Cycle 2)

Unsure of what to do, we brainstormed what might be causing the problem. As we lacked the materials to test all 30 riboswitches with J23100 in triplicate, we looked to another approach. Our team hypothesized that the J23100 promoter in our plasmid was not strong enough and that a different promoter like T7 would exhibit stronger fluorescence. The goal of our system was also to work quickly, so we believed the T7 promoter would help this.

## Build (Cycle 2)

To test that theory, we began the cloning process again to cut out J23100 and ligate in T7. All 30 riboswitches were cloned with T7 promoter.

![](https://static.igem.wiki/teams/4950/wiki/eng3.jpg)

Figure 3: pFTV1 plasmid template with T7 promoter, riboswitch insert, and mRFP1 gene.

## Test (Cycle 2)

In the second round of testing we ran the samples in triplicate with the proper controls. However, even when testing the most promising riboswitches in triplicate, the results showed no significant fluorescence. Interestingly, our consitutive no-riboswitch control with J23100 expressed fluorescence (as expected), but our no-ribowitch control with T7 failed to fluoresce.

## Learn (Cycle 2)

Our team is taking these results into consideration as we continue with the testing. Once we see a strong fluorescence with the pFTV1\_mRFP plasmid with T7, we will cut our the mRFP1 and add in Tre37A.

![](https://static.igem.wiki/teams/4950/wiki/eng4.jpg)

Figure 4: pFTV1 plasmid template with T7 promoter, riboswitch insert, and Tre37A gene.

As we are still engaged in the Learn phase of this cycle, we will step back and look more closely at each part of our testing. The T7 positive control’s failure to fluoresce indicates that something is inherently wrong with our testing. We will return to the Design phase for both the J23100 and T7 riboswitches, reassess our protocols, and rety our tests with new improvements.

## References

[1] Vezeau, G.E., Gadila, L.R. & Salis, H.M. Automated design of protein-binding riboswitches for sensing human biomarkers in a cell-free expression system. Nat Commun 14, 2416 (2023). https://doi.org/10.1038/s41467-023-38098-0

[2] Amalfitano, E., Karlikow, M., Norouzi, M. et al. A glucose meter interface for point-of-care gene circuit-based diagnostics. Nat Commun 12, 724 (2021). https://doi.org/10.1038/s41467-020-20639-6

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## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.
Below is our protocol book turned into a powerpoint presentation.

This is an embedded [Microsoft Office](https://office.com) presentation, powered by [Office](https://office.com/webapps).

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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# Results

## Results

**Characterization:**

Created riboswitches were characterized by cell free TXTL (Transcription-Translation) reaction measuring mRFP fluorescence (584nm/607nm ex/em) with 5nm bandwith every three minutes at 29°C by Tecan Spark microplate reader. Reactions were performed in triplicate and fluorescence at 607nm was averaged for each time point and plotted against time.

![](https://static.igem.wiki/teams/4950/wiki/result1.png)

Figure 1: pFTV1\_mRFP1 positive control plasmid and no DNA

negative control mRFP1 RFU (607nm) plotted against time.

To determine if the TXTL system is working correctly, pFTV1\_mRFP1 positive control plasmid and no DNA control fluorescence were measured versus time (Fig. 1). pFTV1\_mRFP1 control produced expected increase in fluorescence and eventually leveled off, and no DNA control fluorescence at 607 nm excitation remained approximately the same throughout measurements. From this, our TXTL system was determined to work as intended.

![](https://static.igem.wiki/teams/4950/wiki/result2.png)

Figure 2: mRFP1 RFU (607nm) plotted against time in TXTL

reaction with bFGF2 riboswitch In presence of bFGF ligand.

![](https://static.igem.wiki/teams/4950/wiki/result3.png)

Figure 3: mRFP1 RFU (607nm) plotted against time in TXTL

reaction with bFGF2 riboswitch in absence of bFGF ligand.

bFGF2 riboswitch TXTL fluorescence against time in presence of bFGF ligand (Fig. 2) was plotted and compared to the same reaction without bFGF ligand (Fig. 3). Activation ratio of riboswitches in presence of respective ligands can be determined from ligand, no ligand, and positive control TXTL reactions. Further testing is required to accurately characterize bFGF2 and other riboswitch designs.

![](https://static.igem.wiki/teams/4950/wiki/result4.jpg)

Figure 4: NEB5α E. coli cultures with plasmids, in presence

or absence of Thyroxine from left to right: (1) pFTV1\_mRFP1, w/o Thyroxine;

(2) pFTV1\_mRFP1, w/ Thyroxine; (3) pFTV1\_Thyr1\_mRFP1, w/o Thyroxine;

(4) pFTV1\_Thyr1\_mRFP1, w/ Thyroxine.

![](https://static.igem.wiki/teams/4950/wiki/result5.jpg)

Figure 5: NEB5α E. coli cultures pellets with plasmids,

in presence or absence of Thyroxine from left to right:

(1) pFTV1\_mRFP1, w/o Thyroxine; (2) pFTV1\_mRFP1, w/ Thyroxine;

(3) pFTV1\_Thyr1\_mRFP1, w/o Thyroxine; (4) pFTV1\_Thyr1\_mRFP1, w/ Thyroxine.

NEB5α E. coli cells were cultured at 37°C, 300 RPM for 18 hours with either pFTV1\_mRFP1 or pFTV1\_Thyr1\_mRFP1 plasmids, with or without thyroxine present (Fig. 4). Cells were pelleted by centrifugation and relative red color was observed between cells (Fig. 5). Thyroxine exhibits an inhibitory effect against mRNA synthesis, but some mRFP1 synthesis was observed in cells with thyroxine present in culture (cultures 2 and 3). mRFP1 was also present in pFTV1\_Thyr1\_mRFP1 NEB5α cells with thyroxine in culture (culture 4). To effectively characterize, RFU of pFTV1\_Thyr1\_mRFP1 + thyroxine needs to be normalized to pFTV1\_mRFP1 positive control and pFTV1\_Thyr1\_mRFP1 w/ thyroxine in TXTL reaction. Further testing is required to fully characterize thyroxine riboswitches and other riboswitches with ligands that have inhibitory effects on mRNA synthesis.

**Further Work:**
At the time of writing, we ran out of time to obtain additional data due to length of assays and our microplate reader requiring repairs. These factors did not allow us to collect and interpret the amount of data we originally wanted, but we are continuing to perform characterizations and determine riboswitch activation ratios to compare to computational models.

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