Engineering Success | Stony-Brook

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.

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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.

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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.

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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.

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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.

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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.

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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.

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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

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.

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.

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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.

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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.

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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

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.

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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).

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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

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.

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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.