iGEM 2024 — Team stony-brook

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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![](https://static.igem.wiki/teams/5090/home/notebook.jpg)

**Notebook**

*What is our Notebook?*

Our notebook serves as a permanent, chronological record of the scientific work we completed over the last few months as we worked on our iGEM Project. Click below to check out our notebook!

[Our story begins in January 2024...

iGEM 2024
 Notebook](https://static.igem.wiki/teams/5090/neweduimages/team-notebook-1.pdf)

*references*

View References

35+ Amazing CSS Book Effects (Free Code + Demos). (2020). Devsnap.me. https://devsnap.me/css-book-effects

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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[miRNA Heat Shock Assay Optimization](#mirna)
[Human Argonaute 2 Protein](#human)
[Dual Regulation Plasmid](#dual)
[Cell Free System Testing](#cell)

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![](https://static.igem.wiki/teams/5090/home/engineering-wiki.jpg)

**Engineering**

The Engineering Page is essential for documenting the iterative process of designing, building, testing, and learning from experiments. It provides a structured framework that allows teams to systematically improve their synthetic biology projects by identifying challenges and solutions at each stage. This transparency helps other researchers understand the methodology and rationale behind the team's work, fostering collaboration within the iGEM community. We hope this page succeeds in fulfilling those requirements.

For each engineering cycle, we went through the basic 4 steps: Design, Build, Test, Learn.

The cycles are as follows: miRNA Heat Shock Assay Optimization → Human Argonaute 2 Protein → Dual Regulation Plasmid → Cell Free System Testing

*Cycle 1: miRNA Heat Shock Assay Optimization*

*Design*

---

A major question we have for our bacterial-based miRNA detection system is whether bacteria is able to take up miRNA. We could not find any sources that provide documentation on this, so we designed a qualitative assay with the help of our secondary PI, Dr. Gunn.

(1). Structure of miRNA mimics: We first did a literature search and consulted with advisors to determine the specific structure and features of our miRNA. We found that miRNAs generally existed as duplexes in the blood prior to complementary matching (Corey et al., 2016) and were o-methylated (Carré et al., 2019). We chose to order miRNA-326 mimics as an RNA duplex with a FAM fluorophore attached to the passenger strand (the sequence complementary to miRNA-326).

(2). Concentration of miRNA to test: we referenced SASTRA Thanjavur 2019 iGEM and our dry lab modeling of the minimum concentration of miRNA required for our system to work using ODEs. From this, we settled on 1 pM, 10 pM, 100 pM, and 1 nM.

(3). Heat Shock Method: We consulted our Advisor Melanie, who informed us that she has done successful transformations with similar-length double-stranded DNA molecules to our miRNA. We decided to follow her suggestion and prepare our bacteria like how we would for any ordinary transformation, and follow a general heat shock procedure.

*Build*

---

We prepared competent MRE600 E. coli cells using calcium chloride and aliquoted our FAM-miRNA-326 into different concentrations. Then, a heat-shocked transformation was performed with the miRNA and the competent MRE600 cells. Finally, we washed the MRE600 cells two times with SOC and then once in PBS to wash off any miRNA stuck to the membrane of the cells.

*Test*

---

![](https://static.igem.wiki/teams/5090/new-eng/t.png)

Confocal images of MRE600 transformed with FAM-miRNA-326 at different concentrations.

To qualitatively assess if the bacteria uptook miRNA-326, we prepared a slide of our bacteria sample at each concentration of miRNA for confocal microscopy. We took several images of the
bacteria under confocal microscopy and adjusted the images for contrast to assess the level of fluorescence.

*Learn*

---

Our initial experiment yielded a lot of internal noise, indicated by the patches of bright fluorescent patches. In this initial test, the observable difference between the experimental and control groups, particularly 1 nM having more fluorescence than the negative control gives us the implication of the possibility of miRNA being transfected into the bacteria; however, we were worried about the background fluorescent patches and the inconsistent pattern with the level of fluorescent among the groups. Fluorescent levels did not decrease linearly with decreasing concentrations of fluorescently labeled miRNA. We were worried that the miRNA might be stuck to the membrane of our bacteria and not inside the cell.

We hypothesized that we did not perform enough washes with PBS, leading to extraneous fluorescence signals, such as from miRNA caught on the cell membranes. We also hypothesized that our cell density was too high and inconsistent both within and among samples which is not optimal and could lead to inconsistent results.

*Build*

---

Therefore, we repeated this assay and made sure to 1) verify the OD and perform a serial dilution to standardize cell density and 2) add two more PBS washes before imaging the cells using confocal microscopy. We chose to repeat this with the 1 nM group first as this group had the highest level of fluorescence compared to the control group in the initial results.

*Test*

---

We prepared a slide of our bacteria sample at each cell density transfected with 1nM of fluorescent miRNA for confocal microscopy like before. We then also imaged the bacteria under confocal microscopy and adjusted the images for contrast to assess the level of fluorescence.

*Learn*

---

![](https://static.igem.wiki/teams/5090/new-eng/screenshot-2024-10-02-004417.png)

Confocal images, adjusted for contrast, of MRE600 transformed with FAM-miRNA-326 at 1 nM and control group.

In the second imaging of the cells, we found there were fewer background fluorescence signals and clumps of high fluorescence. Therefore, we made adaptations to our heat shock protocol for miRNA. We found the ideal cell density to be at an OD of 0.6 with 5 total washes instead of 3. Our next steps are to experiment with these adjustments with picomolar ranges of miRNA and below to account for general miRNA concentration in blood. Additionally, we would like to test with a higher magnification on the microscope and perhaps quantify the amount of miRNA molecules taken up by the bacteria.

*Cycle 2: Human Argonaute 2 Protein*

*Design*

---

As Argonaute2 is a large protein designed to be expressed in humans, we had to make several adaptations to optimize it for bacterial expression. First, we codon-optimized the sequence of Ago2 to modify some of the rare codons in bacteria. We did this by utilizing the IDT codon optimization tool with the following settings: Sequence Type: DNA, Product Type: gBlocks Gene Fragments, Organism: Escherichia coli. We also decided to add a fusion tag to Ago2 in order to increase its solubility, and subsequently, its expression in bacteria. From our literature search, we found that Ago2 is typically fused with either a GST or an MBP tag. We chose to go with a GST tag as the SBU Glynn lab generously supplied us with GST antibodies. Initial experiments with TEDA cloning showed that smaller DNA fragments typically did not assemble well in bacteria. Thus, we decided to order the sequence of Ago2 in two large fragments from Genscript, with accompanying primers to generate overhangs.

*Build*

---

![](https://static.igem.wiki/teams/5090/eng-img/image7.png)
We utilized TEDA to assemble the fragments into the pJL1 backbone. First, we cloned the codon-optimized Ago2 fragments that included the promoter and terminator region into our plasmid using our competent DH5 alpha cells made in house. Then, we did a sequential assembly to add in the GST tag. For the second assembly, our initial assembly with competent DH5 alpha cells made in-house was unsuccessful, so we attempted the assembly again with NEB-competent DH10B cells, which was successful. In each cloning stage, we confirmed the proper assembly of the fragments through sequencing.

*Test*

---

![](https://static.igem.wiki/teams/5090/eng-img/image9-2.png)

Results of our coomassie staining. Note that there are no distinct bands between 100 and 150 kDa, where our constructs would have been expected to appear outside of the control no plasmid columns.

![](https://static.igem.wiki/teams/5090/eng-img/image1-2.png)

This is our Western Blot. Note that the ladder is the same as indicated in the image for the attempted Coomassie, and that no band appears in the middle column, in which GST-Ago2 was loaded

We attempted to qualitatively assess the expression of Ago2 in bacteria. We transformed pJL1 with Ago2 into MRE600 E. coli cells. First, a starter culture of the bacteria was created with 2mL of LB and appropriate media and incubated overnight at 37 C, 250 rpm. The culture is transferred to 1:100 of fresh LB and then incubated overnight at 25 C, 250 rpm. We then lysed the cell and did a coomassie staining.
We decided to then run a Western blot of Ago2 using antibodies for GST in hopes of better results. However, the Western blot yielded no bands for our samples, which indicated that Ago2 was not expressed.

*Learn*

---

The expression condition of our Ago2 protein still has to be refined. Our coomassie did not show a desired protein band with the correct size and we did not get any bands with our western. Additionally, when we are monitoring our Ago2 bacteria, we noticed that the OD decreases around the 4-hour mark and then slowly increases with several plateaus within the next two hours. This all seems to imply that the constitutive expression of our Ago2 plasmid under 25C and 250rmp might be too metabolically stressful for our bacteria. We hypothesize that it should be expressed at an even lower temperature (15C) after consulting with our advisor Melanie.

For future projects, we now understand the importance of using an inducible promoter in order to control the start of expression to optimize bacteria growth. In addition, a T7 promoter may have been more ideal as opposed to the endogenous Anderson promoters for a large, difficult to express protein such as Ago2, especially in a bacterial context. Given time constraints and reasons discussed further in the engineering cycle for our dual regulation plasmid, we decided to pursue the characterization of Argonaute2 in a cell-free system using our construct that we built in this cycle. This is discussed in our cell-free system cycle, also below.

*Cycle 3: Dual Regulation Plasmid*

*Design*

---

We decided that the dual regulation plasmid would have the combined LacI-P2A-L7Ae construct under the regulation of Ago2 via the miRNA target site, and GFP under the regulation of LacI and L7Ae via two lac operators and a kink turn, respectively. These two major gene fragments would be located on different parts of the plasmid backbone. We decided to use pACYC as the backbone due to its origin of replication, p15A, it fits into the B incompatibility group and thus can be expressed alongside our plasmid for Ago2 (pJL1) which is in incompatibility group A due to its ColE1 ori (Morgan, 2014). Further, we decided to take three different approaches to adapting a previously mammalian-based system into an E. coli-based setting. Specifically, we considered where to place the microRNA target sequence. The three approaches we chose were the following.

(1). Placing the microRNA target site in the open reading frame/coding sequence, buffered by an additional P2A.

(2). Placing the microRNA target site in the open reading frame/coding sequence as a scar on LacI.

(3). Placing the microRNA target site in the 5' UTR, overwriting a later portion of the ribosome binding site.

Prior to cloning, we designed primers to insert our fragments into this backbone. We ordered the “DR” fragment in two large parts about 2000 base pairs long due to constraints from Genscript for its synthesis of fragments any larger than that. We ordered the GFP fragment as a whole single fragment from TWIST.

*Build*

---

We utilized TEDA to assemble the fragments into PACYC. First, we cloned the whole GFP fragment, including two lac operators and the kink turn for its regulation, into our plasmid. We then extracted the plasmid from our cloning vector through miniprep. After confirmation through sequencing seen in the Test section below of a successful cloning, we used the primers for dual-regulation with TEDA to assemble that into the new backbone. We miniprepped again, and sent off to sequencing, the results of which are also seen below. We used sequencing to confirm whether or not cloning was successful. The first plasmid map illustrates the successful cloning of GFP into PACYC. The latter plasmid maps demonstrate successful cloning of two of the dual-regulation system approaches and an issue with the third, the one in which LacI has the microRNA target site as a scar.

*Build*

---

![](https://static.igem.wiki/teams/5090/eng-img/image3.png)

![](https://static.igem.wiki/teams/5090/eng-img/image4.png)

*Build*

---

![](https://static.igem.wiki/teams/5090/eng-img/image6.png)

![](https://static.igem.wiki/teams/5090/eng-img/image8.png)

*Build*

---

Unfortunately, in the proposed plasmid with “T326” as a scar on LacI, a deletion mutation occurred in one of four guanines in a row near the end of T326 and the beginning of the coding sequence for LacI, leaving only three guanines and nearly the whole DR construct thrown off the reading frame. As discussed below, we attempted site directed mutagenesis to reinsert the missing guanine but were unsuccessful as of the time of writing of the wiki. We will continue to attempt to complete this SDM prior to the Jamboree and will update then.

*Test*

---

![](https://static.igem.wiki/teams/5090/eng-img/image10.png)
![](https://static.igem.wiki/teams/5090/eng-img/image5.png)
In terms of GFP expression, we were able to confirm expression through confocal microscopy and cell plate reader. We learned that the optimal cell density for measuring fluorescence systems using E. coli promoters is 0.05. We were unable to find any statistical significance between different cell densities and fluorescence signals.

We wanted to confirm whether or not this cell plate reading was accurate qualitatively so we conducted confocal microscopy, and the results were positive.

*Learn*

---

As a result of this stark contrast between the confocal microscopy results and the cell plate reader under otherwise equal conditions, we suspect that the internal noise of other cellular components disrupts the fluorescence signal. Although we had already planned out text in cell-free systems, the issue of background noise in addition to feedback from stakeholders about the ease of implementation inherent to cell-free systems as opposed to bacterial implementation heightened our emphasis on such testing.

*Cycle 4: Cell Free System Testing*

*Design*

---

The previous results for cycles 2 and 3 (for Ago2 and dual-regulation, respectively) pointed us towards testing in a cell-free system. Though we had planned to do so from the beginning, this heightened our desire to shift our focus. We utilized the same plasmids as had been designed in the earlier engineering cycles (GST-Ago2 on the pJL1 backbone, and DR on pACYC backbone).

*Build*

---

For each cell-free reaction, we set up the cell-free extract based on NEB’s protocol with one slight modification. Due to our use of Anderson promoter J23100, we had to purchase endogenous E. coli RNA polymerase to substitute the T7 RNA polymerase that would have been used if our genes were under T7 promoters. After setting up, we incubated at 37 degrees in the shaking incubator. Our kit from NEB advised us to check results as early as two hours and through up to 10 hours. We then would save the extract for later tests or test according to what was necessary, as see below.

*Test*

---

![](https://static.igem.wiki/teams/5090/eng-img/image11.png)
Our tests began with again confirming whether not GFP alone would express. Based on cell-plate readings, we were able to achieve expression with our GFP plasmid after four hours at an RFU of 300,000, with a plateau after that point. Further testing to evaluate the expression of the DR system and Ago2 is ongoing at the time of writing.

*Learn*

---

For our tests to confirm GFP expression we still experienced background fluorescence from cell free systems expressing pACYC alone and deionized (di) water, we were able to achieve nearly double the RFU of GFP in our cell free system incubated for four hours. This is compared to our previous result in which our cells expressing GFP in the best case had no more than a 25% larger RFU than cells with an empty pACYC backbone. This validated our hypothesis that cell free system expression would be clearer than that in normal cells.

As of the time of writing, characterization of the dual regulation repressors and Ago2 are ongoing.

*references*

View References

(Scroll Inside the white box to scroll through references)
Dimitrova, D. G., Teysset, L., & Carré, C. (2019). RNA 2'-O-Methylation (Nm) Modification in Human Diseases. Genes, 10(2), 117. https://doi.org/10.3390/genes10020117

Matsui, M., Prakash, T. P., & Corey, D. R. (2016). Argonaute 2-dependent Regulation of Gene Expression by Single-stranded miRNA Mimics. Molecular Therapy, 24(5), 946–955. https://doi.org/10.1038/mt.2016.39

Morgan, K. (2014). Plasmids 101: Origin of Replication. Addgene.org. https://blog.addgene.org/plasmid-101-origin-of-replication

Team:SASTRA Thanjavur/Results - 2019.igem.org. (2019). Igem.org. https://2019.igem.org/Team:SASTRA\_Thanjavur/Results

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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[Timeline](#timeline)
[Cloning](#cloning)
[Transformation](#transformation)
[Assays](#assays)

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![](https://static.igem.wiki/teams/5090/home/experiments-new.jpg)

**Experiments**

![](https://static.igem.wiki/teams/5090/exp/1.jpg)

![](https://static.igem.wiki/teams/5090/exp/2.jpg)

![](https://static.igem.wiki/teams/5090/exp/3.jpg)

![](https://static.igem.wiki/teams/5090/exp/4.jpg)

![](https://static.igem.wiki/teams/5090/exp/5.jpg)

![](https://static.igem.wiki/teams/5090/exp/6.jpg)

*Experiments Description*

Scroll down to see a detailed description of all the experiments conducted by our team over the last several months that helped us achieve our results.

*Cloning*

(1). Primers were designed using SnapGene's Gibson assembly feature to contain appropriate 5' overhangs for assembly, and trimmed following Xia et al paper for the correct overhang length and annealing temperature. Annealing Temperature of the primer was calculated using the NEB Tm Calculator. (use buttons below to see full protocols)

(2). Gene Fragments amplification and plasmid linearization were achieved by PCR with NEB Q5 2x master mix following manufacturer's protocol. The PCR reaction mixture is then analyzed on a 1% agarose gel to check for the correct length prior to plasmid assembly. Once we confirmed that the PCR product is the right length, PCR purification was carried out using Thermo Fisher GeneJET PCR purification kit following manufacturer’s protocol.

(3). Gel Extraction: Due to high amounts of nonspecific bands for our Ago2 PCR, we performed a gel extraction using a 1% agarose gel and the GeneJet Gel extraction kit from Thermo Fisher. PCR reaction was scaled up to 100uL, and 25 uL of the reaction mixture was loaded into each lane of the gel. DNA from Exercised gel containing our band of interest was extracted following manufacturer’s protocol, but eluted with 30uL of TE as opposed to 50 uL.

[DNA Gel Electrophoresis Protocol](https://static.igem.wiki/teams/5090/exp-protocols/cloning/dna-gel-electrophoresis-protcol.pdf)
[Q5 PCR Protocol](https://static.igem.wiki/teams/5090/exp-protocols/cloning/q5-pcr-protocol-from-neb.pdf)

(4). Preparation of Competent Cells:

(i). Expression Strain MRE600 or BL21\*:
A 2 mL starter culture of MRE600 or BL21\* is grown overnight at 37 C at 250 rpm with LB. Note that for expression of Ago2, we grew cells at 25 C. The culture is then inoculated the following day in 200mL of fresh LB and grown to an OD600 of 0.35-0.4. The cells are then harvested, centrifuged, and treated with a solution containing HEPES and CaCl2. After that, it is centrifuged and resuspended in a solution containing HEPES, CaCl2, and DMSO. Cells are stored at -80 until use. (see full protocol using the button below)

(ii). Cloning:
For our initial assemblies (Promoter+RBS+GFP+Terminator in PACYC and Promoter+Ago2+Terminator in PJL1) involving products less than 5000 base pairs, we used competent DH5 alpha cells prepared the same way as MRE600 or BL21\* described above. Future assemblies involving larger products failed with our in-house competent DH5 alpha, and we used the DH10B cells provided by NEB.

[CaCl method of making Competent Cells Protocol](https://static.igem.wiki/teams/5090/exp-protocols/cloning/cacl-method-of-making-competent-cells.pdf)

![](https://static.igem.wiki/teams/5090/experiments/image1.png)
(5). T5 exonuclease-dependent assembly:
To assemble all our gene fragments into our plasmid backbone, we used the T5 exonuclease-dependent assembly method from Xia et. al paper because 1) the 2023 SBU iGEM team has had success and 2) for its low cost. This is a modified version of the Gibson assembly where gene fragments and backbones with appropriate overhangs are incubated with just a T5-exonuclease for 30 minutes. The resulting product is then transformed into the bacterium for the rest of the repair (see full protocol using the button below) This can be referred to as TEDA cloning.

[TEDA Cloning Protocol](https://static.igem.wiki/teams/5090/exp-protocols/cloning/teda-cloning-protocol-2024.pdf)

*Transformation*

A standard heat shock transformation is employed (see detailed protocol using the buttons below).

[Agar Plate Protocol](https://static.igem.wiki/teams/5090/exp-protocols/transformation/agar-plate-protocol.pdf)
[LB Media Protocol](https://static.igem.wiki/teams/5090/exp-protocols/transformation/lb-media.pdf)
[Glycerol Stock Protocol](https://static.igem.wiki/teams/5090/exp-protocols/transformation/making-glycerol-stocks.pdf)
[Transformation for MRE 600 Protocol](https://static.igem.wiki/teams/5090/exp-protocols/transformation/transformation-protocol-for-mre-600.pdf)

*Assays*

(1). miRNA Heat shock Assay:

A major question we hoped to answer was whether or not a bacteria-based microRNA detector could be possible, feasible, and efficient. Through our countless literature reviews, we did not find any sources documenting whether it is possible for bacteria to take up miRNA, which is instead endogenous to eukaryotes. Therefore, we set out to design an experiment to qualitatively assess whether bacteria can uptake miRNA.

Briefly, competent E. coli cells prepared chemically with protocol mentioned above are heat-shocked with fluorophore labeled mi-RNA mimic ordered from IDT. These cells are then washed twice with SOC media and then three times with PBS. Samples are then analyzed with confocal.
(See full protocol using button below)

[miRNA Heat Shock Assay Protocol](https://static.igem.wiki/teams/5090/exp-protocols/assays/mirna-heat-shock-assay.pdf)

(2). Fluorescence Plate Reader Readings:

(i). GFP Calibration Curve

A GFP Calibration curve was generated following iGEM's Plate Reader Fluorescence Calibration protocol (but with GFP protein).We obtained wildtype green fluorescent protein from SBU Karzai lab, and checked for its concentration using the nanodrop.

(ii). Fluorescent Reading of Bacterial Sample

5mL of bacterial samples were inoculated overnight at 37C and 250 rpm in LB supplemented with the appropriate antibiotics. The next day, cells are harvested and diluted to an OD below 1 for OD600 reading, washed three times in PBS, and resuspended in the appropriate amount of PBS to ensure the same cell density among all experimental and control samples. (See our full protocol using buttons below)

(iii). Fluorescent Reading of Cell-Free System

Cell-Free systems were set up using NEBExpress® Cell-free E. coli Protein Synthesis System with the T7 polymerase provided with the kit substituted with endogenous e.coli polymerase saturated with S70 also purchased from NEB. The final reaction mixtures were incubated for four hours at 37 C at 250rpm. For reactions assessing functionality of our full circuit and target miRNA (miR-326), 1 nmole ofmiR-326 mimics ordered from IDT were added before incubation along with 1uL of 2uM MgCl2 solution.

[NEB Cell-Free System Preparation and Categorization Protocol](https://static.igem.wiki/teams/5090/exp-protocols/assays/cell-free-system-preparation-and-categorization.pdf)
[Fluorescence Quantifying Assay For Bacteria Protocol](https://static.igem.wiki/teams/5090/exp-protocols/assays/fluorescence-quantifying-assay-for-bacteria.pdf)
[Microscopy agarose gel slide mounting Protocol](https://static.igem.wiki/teams/5090/exp-protocols/assays/microscopy-agarose-gel-slide-mounting.pdf)

(3). Western:
The N-terminal of our Human Argonaute2 protein (Ago2) was fused to glutathione S-transferase. Because our protein is under an endogenous promoter, we opted for a western blot as the expression level of our protein cannot be identified with staining methods such as a coomassie staining.

Due to the limit of time, we were only able to test expression of our argonaute protein in MRE600. Briefly, a 2mL starter culture was inoculated overnight at 37 C and 250 rpm. The following morning, this starter culture is transferred to 200 mL of fresh LB, and grown at 25 C for 4hr, 8hr, and overnight.

Extracted protein samples are separated by SDS-PAGE and transferred to a nitrocellulose membrane by semi-dry electroblotting. Primary anti-GST Tag antibodies and a secondary peroxidase-conjugated anti-goat antibody were both kindly given by SBU Glynn Lab. Bound antibodies were visualized by an enhanced chemiluminescence system (ECL) according to the manufacturer's protocol (Thermofisher).

[SDS page gel protocol](https://static.igem.wiki/teams/5090/exp-protocols/western/sds-page-gel-protocol-from-sbu-gunn-lab.pdf)
[Western Blot Protocol](https://static.igem.wiki/teams/5090/exp-protocols/western/western-blot-protocol-from-sbu-gunn-lab.pdf)

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{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block lead
%}Description of our results and future plans{% endblock %} {% block scrollspy
%}

[TEDA - Gel Electrophoresis](#gel)
[TEDA - Sequencing](#cloneseq)
[Confocal](#cnfc)
[Plate Reader](#cprr)
[Coomassie](#cmss)
[Western](#wstb)
[Conclusion](#pfrc)

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![](https://static.igem.wiki/teams/5090/neweduimages/results-item.jpg)

**Results**

*TEDA - Gel Electrophoresis*

Below is a selection of gels relevant to our cloning, with
explanations for what each meant. This includes all of our fragments,
linearized plasmid backbones, and linearized plasmids that are
partially assembled. Note that our PCR reactions have resulted in specific bands within the target length (except for our Ago2 fragments which we had to perform a gel extraction).

*Dual Regulation*

---

![](https://static.igem.wiki/teams/5090/results/results-gel-dr-1.png)

*GST-Argonaute2*

---

![](https://static.igem.wiki/teams/5090/results/results-gel-ago2-1.png)
From left to right we have linearized PJL1 plasmid backbone (X2),
Non-codon optimized Ago2 fragment (with promoter, ribosome binding
site (RBS), and terminator sequence), first half of codon optimized
Ago2 fragment (with promoter and ribosome binding site), second half
of codon optimized Ago2 fragment (with terminator), MBP tag
fragment, all with overhangs.

*More Repressor fragments + Ago2 phase 2 fragments*

---

![](https://static.igem.wiki/teams/5090/results/results-gel-ago2-2-new.png)

From left to right we have repressor fragment (microRNA target site in the 5' UTR, overwriting a later portion of the ribosome binding site), first half of repressor fragment (microRNA target site in the open reading frame/coding sequence, buffered by an additional P2A), second half of repressor fragment (microRNA target site in the open reading frame/coding sequence), linearized assembled PJL1 plasmid with codon optimized Ago2, GST tag fragment, all with overhangs.

*TEDA - Cloning and Sequencing Results*

For each plasmid we cloned using TEDA or attempted to edit using site-directed mutagenesis, we would then purify that plasmid from the cloning vector and send it off to sequencing. We did this to gain confirmation on whether the cloning was successful or not. Below is a selection of the plasmids we sent off the sequencing and related commentary, including indication of whether we went on to use a given plasmid in future experiments.

*Dual Regulation Plasmid*

---

![](https://static.igem.wiki/teams/5090/results/results-dr-plasmid-2.png)
![](https://static.igem.wiki/teams/5090/results/results-dr-plasmid-1.png)
1. GFP Ordered in Small Fragments: Initial attempts, in which we attempted to assemble several small fragments, including a separate promoter, RBS+coding sequence, and terminator part, were unsuccessful. The plasmid did not uptake the small gene fragments.

2. GFP Whole Fragment: Primers were designed to insert a newly ordered whole GFP fragment, from promoter to terminator, into the pACYC backbone. Unlike the previous attempts with smaller fragments, this was successful in creating the GFP plasmid that we would go on to use for later experiments as the “control” for normal GFP expression.

3. Insertion of Dual-Regulation System: Primers were then designed to insert sequences for all three proposed versions of the dual-regulation system, this time also all designed from promoter to terminator to avoid the issues encountered with cloning small fragments in TEDA. The three plasmids show that the fragments were successfully cloned into the new GFP-pACYC backbones. This occurred with no issues for DR with the target site in the 5’ UTR and DR with the target site in the ORF followed by P2A. We are conducting characterization of those two plasmids and hope to present more information than is currently provided at the Jamboree. However, in our plasmid into which we cloned DR with the target site as a scar on LacI, there was a point mutation. Specifically, a deletion occurred of one guanine in a series of four guanines that constituted a region of overlap between the target site and the LacI coding sequence. As a result, the open reading frame for nearly the whole DR construct was displaced. We went on to test the former two DR plasmids, and attempted to correct the latter with site-directed mutagenesis.

See the images below for the three plasmids.

![](https://static.igem.wiki/teams/5090/results/results-dr-plasmid-5.png)

![](https://static.igem.wiki/teams/5090/results/results-dr-plasmid-4.png)

![](https://static.igem.wiki/teams/5090/results/results-dr-plasmid-3.png)

The three plasmids show that the fragments were successfully cloned into the new GFP-pACYC backbones. This occurred with no issues for DR with the target site in the 5’ UTR and DR with the target site in the ORF followed by P2A. We are conducting characterization of these two plasmids and hope to present more information than is currently provided at the Jamboree. However, in our plasmid into which we cloned DR with the target site as a scar on LacI, there was a point mutation. Specifically, a deletion occurred of one guanine in a series of four guanines that constituted a region of overlap between the target site and the LacI coding sequence. As a result, the open reading frame for nearly the whole DR construct was displaced. We went on to test the former two DR plasmids, and attempted to correct the latter with site-directed mutagenesis.

4. Site Directed Mutagenesis: We designed primers meant to re-add the missing guanine into the plasmid with DR with the target site as a scar. However, these mutagenies failed. Plasmidsaurus was unable to sequence any of the plasmids we purified from cells that were transformed with the PCR products of SDM. We considered it is possible due to higher-than-preferred annealing temperature that the SDM failed. Attempts to redo this SDM with different primers with more appropriate annealing temperatures are ongoing.

*Ago2 Plasmid*

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![](https://static.igem.wiki/teams/5090/results/results-ago2-plasmid-2.jpg)

*Confocal Microscopy*

As we hope to detect microRNA concentrations in patients'
bloodstream, we have to consider pertinent regulations regarding
handling of blood. The United States Food and Drug Administration
(FDA) maintains several pertinent regulations with regards to blood
collection, preparation, storage, transportation, and testing. Upon
review of these regulations, we found that outside of the blood
handling, storage, transportation, and disposal performed by
clinicians, our system will need to conform with FDA regulations
regarding In Vitro Diagnostic products. This includes both for our
actual cell-free system once it receives microRNA, as well as the
microfluidic assay from which the miRNA will be extracted from the
blood.

*Initial miRNA Uptake Trial*

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*Troubleshooting*

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We hypothesized that the internal noise might have been caused by the tagged miRNA adhering to the external membrane of the cells rather than entering them. The inconsistency in the scaling between fluorescence and miRNA concentration could have resulted from variations in cell concentration between samples. To address the first issue, we decided to thoroughly clean the samples by adding two additional PBS washes to eliminate any extraneous fluorescent signals. To ensure consistent cell counts across samples, we verified optical density using a plate reader and conducted serial dilutions of the known optical density samples to standardize the cell concentration per sample.

*Successful miRNA Uptake Trial*

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![](https://static.igem.wiki/teams/5090/results/results-ago2-confocal-2.png)
After implementing the changes identified during troubleshooting, we conducted a second miRNA uptake trial using 100x optical magnification. This trial included a slide with 1 nM of miRNA and a control with no miRNA. The results showed significantly less internal noise, suggesting that the additional washing steps improved the accuracy of the miRNA uptake imaging. The images clearly demonstrate that miRNA at a 1 nM concentration successfully enters our cell strain. Further experiments with higher confocal magnification and picomolar miRNA concentrations could provide additional support for our hypothesis that blood-level concentrations of miRNA can effectively enter and interact with our cell system.

*GFP Production*

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![](https://static.igem.wiki/teams/5090/results/results-ago2-confocal-3.png)
Although we successfully generated quantitative data for GFP fluorescence, we opted to confirm these results qualitatively, as this aligns with how the test is likely to be performed in practice. The confocal microscopy results were quite clear, showing minimal internal noise and a distinct difference in fluorescence between the GFP-producing plasmid and the bacteria without the plasmid. These findings are promising for the future implementation of Micronaut as a qualitative test. We will try to amplify fluorescence signals through testing other variants such as sfGFP. By doing so, we hope to reduce the difficulty of fluorescence measurement in our system so that it requires minimal imaging equipment.

*Cell Plate Reader Results*

To test the efficacy of our system, we quantified the fluorescence emitted under various conditions: Nothing, empty PACYC backbone, PACYC plasmid with just promoter+RBS+GFP+terminator, and PACYC plasmid with GFP and the dual repressors (LacI and L7Ae). Due to limitations to our equipment, we can only measure fluorescence emitted with an excitation wavelength of 485 and emission wavelength of 535 (as opposed to the ideal wavelengths of 475/509) (To test the efficacy of our system, we quantified the fluorescence emitted under various conditions: Nothing, empty PACYC backbone, PACYC plasmid with just promoter+RBS+GFP+terminator, and PACYC plasmid with GFP and the dual repressors (LacI and L7Ae). Due to limitations to our equipment, we can only measure fluorescence emitted with an excitation wavelength of 485 and emission wavelength of 535 (as opposed to the ideal wavelengths of 475/509) (Bauer et al., 1996).

*1. Categorization in Bacteria*

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![](https://static.igem.wiki/teams/5090/results/results-cpr-bac-1.png)
Our first initial measurement of fluorescence yielded unexpected results as our negative control group (bacteria with no plasmid transformed or just an empty PACYC backbone with no GFP protein) had higher fluorescence than bacteria transformed with our plasmid containing just GFP (groups A,B, and C). We think that this is due to the cell density of the bacteria being too high, and signals being blocked off and not properly read by the plate reader. Thus, we performed a serial dilution of the above colonies to find the optimum cell density to measure fluorescence within our system.

![](https://static.igem.wiki/teams/5090/results/results-cpr-bac-2.png)

![](https://static.igem.wiki/teams/5090/results/results-cpr-bac-3.png)

![](https://static.igem.wiki/teams/5090/results/results-cpr-bac-4.png)

![](https://static.igem.wiki/teams/5090/results/results-cpr-bac-5.png)

![](https://static.igem.wiki/teams/5090/results/results-cpr-bac-6.png)

![](https://static.igem.wiki/teams/5090/results/results-ago2-confocal-3.png)

*2. Categorization in Cell-Free System*

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![](https://static.igem.wiki/teams/5090/results/results-cpr-cfs-1.png)

![](https://static.igem.wiki/teams/5090/results/results-cpr-cfs-2.png)

![](https://static.igem.wiki/teams/5090/results/results-cpr-cfs-3.png)

*Coomassie Stains*

After the successful assembly of codon-optimized Ago2 with a GST tag into pJL1 plasmid using TEDA, the plasmid was successfully transformed into MRE600 E. coli cells. Colonies of MRE600 cells were inoculated and grown at 25 degrees Celsius overnight. A coomassie stain was done to confirm the protein expression of Ago2 in the MRE600 cells.

![](https://static.igem.wiki/teams/5090/results/results-coomassie.png)
The Coomassie stain indicated that Ago2 was not being expressed in the cells. There was no band between the expected range of 100-150 kDa in any of the negative controls or experimental groups. We hypothesized that Ago2 was not being expressed because constitutive endogenous expression was not enough for a band to show up with just staining. Additionally, coomassie staining is not specific/precise enough to detect Ago2 (as opposed to a Western blot) as we only know the size of each protein from the staining.

*Western Blot*

![](https://static.igem.wiki/teams/5090/results/results-western.png)
Following the results of the coomassie stain, a Western blot was done using the same frozen lysate of MRE600 cells with the Ago2-GST plasmid. Primary antibodies against GST was used with secondary antibodies against those primary antibodies.

The Western blot confirmed the results of the coomassie stain. No band showed up for both experimental groups of Ago2-GST. This suggests that the expression condition of our Ago2 protein within our bacteria needs further optimization for proper expression.

*Conclusion - Proof of Concept*

When we developed our project proposal, we hoped to create a system that could detect miRNA using fluorescence as a method of diagnosing B-cell lymphoma. We set out three goals for our system, and below is a synthesis of these goals and our progress towards them.

*1. Without any repression, GFP expresses:*

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![](https://static.igem.wiki/teams/5090/results/results-ago2-confocal-3.png)
Our preliminary results indicate that there is a fluorescence signal in both bacteria and cell-free systems expressing GFP under the endogenous e.coli promoter. Further testing and troubleshooting needs to be done in regards to the high background we observe.

*2. With DR, GFP no longer expresses.*

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![](https://static.igem.wiki/teams/5090/results/results-cpr-cfs-2.png)
Fluorescence signal decreases significantly with the addition of dual repressors as indicated by the plate reader results. We were only able to test one of three approaches we designed. Preliminary cell-plate reader evidence shown above also shows recovery of expression after Ago2, suggesting a complete circuit.

*3. With Ago2 and miRNA-326, GFP expresses once again*

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![](https://static.igem.wiki/teams/5090/results/results-western.png)
Our preliminary attempts at expressing Ago2 within our bacteria system depicts that further optimizations are needed. As we know the gene was successfully cloned into the expression vector, we would like to try different conditions such as a lower temperature and longer incubation time to acheive expression. We would also like to perform a Western Blot for Ago2 produced in the S30 cell-free system to observe whether or not Ago2 expression is sucecssful in that context.

*Overall*

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Our preliminary results illustrate our system's potential to be a functional screening test, but equipment such as a plate reader is needed in its current form. In our discussion with various stakeholders, a common recurring theme is the importance of our system being easy to use and having low equipment requirements. We wish to remove the need of this expensive equipment through the following changes that will amplify our fluorescence signal:

1. Switching from endogenous e.coli promoter to T7 Promoter.

2. Using other GFP variants such as sfGFP, or other genes whose expression is visible to the naked eye.

3. Implementation in a paper-based cell-free system, as proposed by 2022 ICJFLS iGEM Team for their miRNA-detecting system ("Proposed Implementation", 2019).

*references*

View References

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