iGEM 2018 — Team Hong_Kong-CUHK

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# Team:Hong Kong-CUHK/Notebook

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RNA Aptamer Probe Influenza Detector

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Email:

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Address:

CUHK, Sha Tin, NT, Hong Kong

CUHK Igem 2018

# Notebook

### Sep 2017 - May 2018

Topic Brainstorming: *Produce acrylamide hydrolase in E. coli, Use docked His-tags to remove heavy metal in sewage, Alter Streptococcus gordonii for oral probiotics, Using Arthrospira platensis to produce oral vaccine, Improvement of toehold switches, Produce a switchable Spinach aptamer.*

Our initial project focused on *Alter Streptococcus gordonii for oral probiotics*. After several meetings with Professors, we decided to focus on *Improvement of toehold switches* and *Produce a switchable Spinach aptamer.*

### Week 1: 29 Apr - 5 May

Frederick designed primers to make several improvements on the original toehold switch design.

Rebecca designed primers for expression of Spinach, tBroccoli, tdBroccoli and Split-Broccoli in E. coli.

### Week 2: 6 May - 12 May

Primers have arrived.

Frederick started reconstructing the toehold switch expression plasmids, however contamination on plates are abundant. He started to eliminate the cause by sanitization of the incubator, shaker, and fridge.

### Week 3: 13 May - 19 May

Frederick, Peter and Elsa are collaborating for cloning of the toehold switch expression plasmids. Frederick noticed that the cloning steps can be reduced a lot by using very long primers for overlap PCR. However, the cloning plan is unchanged.

Contamination is still noticeably abundant.

### Week 4: 20 May - 26 May

Rebecca started cloning the aptamers. She also encountered the contamination issue. She streaked plates for the competent cells and found out that 2 out of 4 batches have yeast-like contamination. Peter and Frederick made some new competent cells.

Using unconfirmed clones for fluorescence assay led to failure. The cause of this is later discovered as possibly the low pH of the M9 medium stock.

### Week 5: 27 May - 2 June

Cloning of the toehold switch plasmids kept facing failure. Sequencing results showed some fragment could not be detected. Gel electrophoresis showed that the pACYCDuet-1-based trigger plasmid has probably undergone homologous recombination. The RFP-expressing switch plasmid is showing very dim light. Primers are designed for changing the promoter from J23100 to the sfGFP promoter.

### Week 6: 3 June - 9 June

Rebecca was trying to use a “Prevent correct folding” method for the Spinach aptamer.

Frederick discovered a paper using “Destabilize folding” method, which he designed primers for expressing the probes in E. coli. The target RNA is the AmpR gene, as it already existed in pSB1A3.

No clones for the changing promoter.

### Week 7: 10 June - 16 June

Cloning of the AmpR probe to pCOLA-J23100 was successful. Clones for changing promoter had dim light at first but bright light after several days in the fridge.

### Week 8: 17 June - 23 June

pCOLA-J23100-AmpR probe and pSB1A3 are co-transformed into DH5alpha for the assay. However, there is no visible difference between the untransformed and the co-transformed cells. Frederick concluded that the expression level of AmpR is too low. The cause of this is later discovered as probably the low pH of the M9 medium stock.

### Week 9: 24 June - 30 June

InterLab duty was assigned to Daniel and Peter.

Frederick transformed the biobricks for cell assays.

### Week 10: 1 July - 7 July

Professors were urging us to have actual progress. Rebecca, Elsa, and Frederick decided to try the “Destabilizing folding” approach and designed probes for hemagglutinin and neuraminidase genes from influenza A. We decided to design them as siRNA and use the data to design better probes. Primers for E. coli expression and in vitro transcription were ordered.

### Week 11: 8 July - 14 July

In vitro transcription kits were bought. Frederick decided to try E. coli expression first. Cloning of most probes were successful, although some had single base mutations.

InterLab study was finished within the week.

### Week 12: 15 July - 22 July

The plasmids for probes and targets were transformed into E. coli C41(DE3) for screening. However, both the probe+target clones and the positive control clones did not have fluorescence. The reason was concluded to be that the ribosome binding sites in the Duet vectors were not removed, causing unfolding of the aptamers. Primers are designed for adding the T7 promoter right before the aptamer or target, which can also be used for templates for in vitro transcription.

### Week 13: 23 July - 30 July

In vitro transcription kit arrived. In vitro transcription using linearized plasmids as templates resulted in very low yield.

### Week 14: 31 July - 5 Aug

Primers for direct T7 promoter has arrived. Cloning of the probes had once again begun.

### Week 15: 6 Aug - 12 Aug

Elsa and Frederick formed the assay cloning team, for cloning of the probes and targets into pRSFDuet-1 based plasmids for assay; Jenny, Daniel and Peter has formed the standard biobrick cloning team for cloning of the basic biobrick probes and composite biobrick T7 Promoter-Probe-T7 terminators.

### Week 16: 13 Aug - 19 Aug

Expression test of the positive control aptamer confirmed that BL21, 37C incubation results in the maximum fluorescence.

### Week 17: 20 Aug - 26 Aug

Probes with the new design were cloned and co-transformed with the target with the old design. After induction, it seems that all probes did not have significantly increased fluorescence with target expression. However, one of the N9 probes and one of the N2 probes seemed to have a little increase. Frederick decided to perform in vitro transcription for those probes using PCR product.

### Week 18: 27 Aug - 2 Sep

Elsa performed the RNA fluorescent assay of probes for H3, H7, N2, N9, and after data analysis, we found that N2 and N9 have statistically difference between probe-target complex and probe only.

We received the BL21 Star (DE3) strain from the NUS iGEM 2018 team.

### Week 19: 3 - 9 Sep

To see whether our aptamer system can perform well after nasal fluid is added, Elsa performed RNA fluorescent assay of N2 and N9 probes in different salt concentration (i.e. potassium ion, magnesium ion, calcium ion and sodium ion) with reference to the salt composition of nasal fluid.

To identify the minimum amount of target needed to have a significant positive result. Elsa also performed the limit of detection assay of N2 and N9 probe, by setting reaction of 2uM of probe with different concentration of target (i.e. 1.5uM, 1uM, 0.5uM, 0.2uM, 0.05uM) and addition of same amount of aptamer folding buffer and fluorogen DFHBI. We found that at least 0.2uM and 0.05uM were required for N9 and N2 probe-target complex to have significant on-off ratio.

To see whether the aptamers can give sufficient fluorescence in household setting, Elsa performed RNA fluorescent assay of N2 and N9 probes with condition of (1) 90 degree Celsius 5 minutes folding time, followed by room temperature 45 minutes incubation time; and (2) room temperature all the way the assay. When compared the result to the protocol (90 degree Celsius 5 minutes folding time followed by 37 degree Celsius 45 minutes incubation time), condition (1) gave a significant on-off ratio, though the fluorescence given were lower than the protocol.

New aptamers were designed and primers needed were ordered.

To see if the E. coli expression results were due to its low expression level, Frederick performed total RNA extraction with the RNA-Snap protocol and tested as the in vitro transcription probes. It seemed that the positive control has a 7-fold fluorescence recovery after RNA extraction, showing the E. coli did not have the optimal environment for the optimal folding of the aptamers. Probes did not have better performance after RNA extraction, we hypothesized that this might be due to misfolding due to the T7 terminator addition.

Primers for Improvement of a previous biobrick was ordered.

Daniel performed transformation of :RAPID-N9, RAPID-N2,T7 promoter-RAPID-N9-T7 terminator, T7 promoter-RAPID-N2-T7 terminator and iSpinach.

### Week 20: 10 - 16 Sep

Primers for previous biobricks improvement and primer for new set of aptamers were arrived. Frederick performed in vitro transcription for those probes using PCR product.

Daniel, Peter and Jenny performed Miniprep for the successful clones: T7 promoter-RAPID-N9-T7 terminator (1 success), T7 promoter-RAPID-N2-T7 terminator (3 successes), RAPID-N9(2 successes) ,checked clone by restriction enzyme digestion.

They also performed ligation and transformation of RAPID-N2, miniSpinach(P1-a5-b3), T7 promoter-miniSpinach-T7 terminator, iSpinach and colony PCR of RAPID-N2(2 successes), miniSpinach(P1-a5-b3)(1 success), T7 promoter-miniSpinach-T7 terminator(1 success), iSpinach(3 successes).They did minipreparation for the successful clones and sent to sequencing.

### Week 21: 17 - 23 Sep

Elsa performed RNA fluorescent assay of probes for H1, H3, H7, N1, N2, N9 and polymerase basic 2. For probes for H3, H7, N2, N9 and polymerase basic 2, 1 among 4 probes tested for each type of surface protein had a ‘good’ on-off ratio.

### Week 23: 1 - 7 Oct

Frederick and Elsa performed real-time pcr of N2 and N9, using DFHBI as fluorogen, to check for the time and optimal temperature for signal development. We found that signal develop started when the temperature reached 75 degrees Celsius on top of 95 degree Celsius 5 minutes folding time, while 20 degree Celsius was the optimal temperature for aptamer to give fluorescence.

### Week 24: 8 - 14 Oct

Frederick and Elsa performed RNA fluorescent assay by mixing probes with targets from different probes to test the specificity of our RNA aptamer Probe. It was found that our probe is orthogonal and specific.


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# Team:Hong Kong-CUHK/Experiments

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RNA Aptamer Probe Influenza Detector

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Email:

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CUHK Igem 2018

# Experiments

### Design Principle of RNA Aptamer Probe

![figure1](https://static.igem.org/mediawiki/2018/2/22/T--Hong_Kong-CUHK--exp_fig1_large.png)
The Spinach aptamer, along with its structural characteristics and photophysics are well-characterized. According to the crystal structures of Spinach and iSpinach-D5, the Spinach aptamer generally consists of two arms, P1 and P2, surrounding a G-quadruplex containing docking site of its fluorogen, DFHBI. While the docking site is indispensable for the formation of the Spinach-DFHBI complex, the lengths of the arms have shown to be less important by previous mutagenic studies to shorten the arms. In particular, the P1 arm length has a dramatic effect on the fluorescence level, which the single-base deletion can lead to complete loss of fluorescence in E. coli.

By adding a 11-bp arm to each side of the P1-truncated Spinach aptamer, which its sequence is complementary to a specific sequence of RNA, it can become a split light-up RNA aptamer with a modulable sequence design targeting influenza RNA. It works by having a misfolded / unfolded conformation of the Spinach aptamer on its own, but changing to a correctly folded conformation of the Spinach aptamer when hybridized to the influenza RNA. (Fig. Right)

### In silico Design of RNA Aptamer Probe

![figure2](https://static.igem.org/mediawiki/2018/a/a3/T--Hong_Kong-CUHK--exp_fig2.png)
As there are no known algorithms for predicting the binding of RNA to DFHBI and the resulting fluorescence level. According to previous data, shortening of the P2 arm did not have significant change in the fluorescence level of the Spinach aptamer. Based on these data, we have made 2 assumptions in our design: (1) The formation of the docking site is dependent on the correct folding of the P1 arm. (2) The P1 arm folding is optimized when the hybridization of the variable regions is the most favorable. Based on these two assumptions, we utilized the Invitrogen BLOCK-iT siRNA Designer, which can find the region of RNA with the least amount of secondary structures, as well as human genome BLAST. The web page generates the sequences of 25 bp length.

The gene sequences of influenza A were chosen as follows (Table Below). The hemagglutinin and neuraminidase genes were selected for influenza subtyping, while the region of Polymerase Basic 2 gene that is ubiquitous in most influenza A genomes were chosen for influenza detection (This was inspired by the influenza expert that we interviewed, [Prof. Lee Shui-shan](https://2018.igem.org/Team%3AHong_Kong-CUHK/Human_Practices)). After inputting the selected sequences in BLOCK-iT designer, we have chosen the ones with the GC content around ~50% and randomly selected a 22 bp region for probe design (Fig. Above).

![table1](https://static.igem.org/mediawiki/2018/e/e4/T--Hong_Kong-CUHK--exp_table1.png)

### *In vitro* screening of probes

##### 1. Production of RNA Aptamer Probes and Target RNA

To produce RNA probes and their target RNAs for assays, we used in vitro transcription kits. We designed oligos that can hybridize to each other and extend in a PCR reaction to overcome to length limit of oligo synthesis. After Phusion PCR, the product was gel purified and used as a template in NEB HiScribe T7 Quick High Yield RNA Synthesis Kit. After DNaseI treatment, the reaction was directly purified using phenol:chloroform extraction, and the RNA concentration was measured with NanoDrop 2000. RNA molar concentration was calculated using the corresponding MW according to its length.

![figure-invitro](https://static.igem.org/mediawiki/2018/e/ee/T--Hong_Kong-CUHK--invitro.png)

##### 2. Aptamer Refolding Assay

In order to investigate the effectiveness of our designed aptamers, in vitro transcribed RNA aptamers (1uM) were mixed with its target, 2X aptamer folding buffer (20mM Tris-HCl, 200mM KCl, 10mM MgCl2) and fluorophore DFHBI, inspired by Kikuchi N, 2016. Controls were set up by mixing the above components without RNA aptamer. The fluorescent signal was observed under blue light box / Chemidoc and was determined by CLARIOstar plate reader.

### Characterization of Aptamer

##### 1. Specificity

Though our aptamers were designed to target hemagglutinin or neuraminidase genes of influenza subtypes, unwanted binding between aptamer and target of two subtypes may happen in real life.
5 of our aptamers (i.e. for N9, N2, H7, H3, PB2) that performed well in previous RNA fluorescence assay were selected to investigate their cross-reactivity. They were mixed with targets from different subtypes sequence and subjected to aptamer refolding assay.

##### 2. Detection Limit

It is important to know the minimum amount needed to distinguish the fluorescence signal under the blue light box by naked eye, in order to understand at which stage of influenza latency can be viral RNA be detected. We have mixed 2uM aptamer (N2-694 and N9-545 aptamers) with different concentration of its target (i.e.1.5uM, 1uM, 0.5uM, 0.2uM, 0.1uM and 0.05uM) and performed aptamer refolding assay as usual.

##### 3. Ion Dependency

As the test sample will be individuals’ nasal fluid, we wondered if the addition of nasal fluid will influence the RNA aptamer folding, for instance, the ionic composition in nasal fluid.
We have selected sodium ions, potassium ions, calcium ions and magnesium ions in different concentration to test for the fluorescent signals given from our 2 probes (N2-694 and N9-545 aptamers). Ions were added to aptamer folding buffer in form of salt solution.
Concentration of different ions in nasal fluid, in aptamer folding buffer are shown below.

![figure-invitro](https://static.igem.org/mediawiki/2018/2/26/T--Hong_Kong-CUHK--Ion_Buffer_List1.png)

The range of concentrations selected for each ions are shown below. Those red number indicate the original amount of ions presented in the aptamer folding buffer.

![figure-invitro](https://static.igem.org/mediawiki/2018/7/79/T--Hong_Kong-CUHK--Ion_Buffer_List.png)

### Optimization of Aptamer Folding Condition

In order to trace the change in fluorescence signal with the time and temperature, BioRad real-time PCR machine was employed for the monitoring fluorescence signal during the aptamer refolding assay using SYBR Green Mode.

### E. coli Expression of RNA Aptamer Probes

Although our project aims to produce a probe outside the context of a cell, the screening of probes is cheaper if they can be expressed and tested in E. coli. Moreover, it would allow our future iGEMers to be able to use our probes in their usual contexts, which is in a chassis like E. coli. To test if this is applicable, we co-expressed the probes and targets using the Novagen Duet vector system. While we modified pRSFDuet-1 vector to generate a T7 promoter-based aptamer expression system, we also cloned the target genes into the multiple cloning site of pACYCDuet-1. The resulting constructs are as follows (Fig. Below).

![table1](https://static.igem.org/mediawiki/2018/9/9e/T--Hong_Kong-CUHK--exp_fig3.png)

While the RNA aptamer probes are immediately following the T7 promoter and followed by the T7 terminator, the influenza RNA is included in the transcripts of the MCS. After IPTG induction in co-transformed BL21 Star (DE3), they are incubated in medium containing DFHBI and their fluorescence in measured by BMG CLARIOStar microplate reader. We obtained the strain from Team NUS Singapore-A as a part of our [collaboration](https://2018.igem.org/Team%3AHong_Kong-CUHK/Collaborations).

### Improving existing biobricks and project: RNA Reporters for Promoter Activity Measurement

Previously, the Technical University of Denmark 2014 team utilized the tRNALys3-Spinach2.1 (BBa\_K1330000) for measurement of the Anderson promoter library. Due to the illegal site of the Spinach2 sequence, they designed the Spinach2.1 RNA Reporter which has lower fluorescence than the original Spinach2.

Despite the brightness of Spinach2, It has a melting temperature of 38°C. This means the characterization of heat-induced promoters is not favored by using this reporter. On the other hand, our collaborating partner NUS iGEM 2018 is trying to develop a RNA reporter connected with the chaperone promoter phtpG.

To overcome the obstacle of (1) brightness and (2) temperature sensitivity, we constructed 3 new RNA aptamers for the iGEM registry, all using the tRNALys3 scaffold of the original biobrick. The biobricks include miniSpinach, iSpinach and dimeric Broccoli. Their in vivo fluorescence are tested in E. coli.

### References

1. W. Q. Ong, Y. R. Citron, S. Sekine, and B. Huang, “Live Cell imaging of endogenous mRNA Using RNA-Based fluorescence ‘turn-on’ probe,” ACS Chem. Biol., vol. 12, no. 1, pp. 200–205, 2017.
2. BLOCK-iT Designer. Invitrogen. https://rnaidesigner.thermofisher.com/rnaiexpress/.
3. K. K. Alam, K. D. Tawiah, M. F. Lichte, D. Porciani, and D. H. Burke, “A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly in Vivo,” ACS Synth. Biol., vol. 6, no. 9, pp. 1710–1721, 2017.
4. Burke W. The Ionic Composition of Nasal Fluid and Its Function. Health [Internet]. 2014 Mar 6:720-728. Available from:
   http://dx.doi.org/10.4236/health.2014.68093
5. Kikuchi N, Kolpashchikov DM. Split Spinach Aptamer for Highly Selective Recognition of DNA and RNA at Ambient Temperatures. ChemBioChem [Internet]. 2016 Jul 15;17(17):1589–92. Available from: http://dx.doi.org/10.1002/cbic.201600323

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# Team:Hong Kong-CUHK/Results

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RNA Aptamer Probe Influenza Detector

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Email:

igem18.cu@gmail.com

Address:

CUHK, Sha Tin, NT, Hong Kong

CUHK Igem 2018

# Results

### 1. *in vitro* Probe Screening

#### Aptamer Folding Assay

After generating 4 aptamers with BLOCK-iT RNAi Designer for H1, H3, H7, N1, N2, N9, Polymerase Basic 2 (PB2) each, we performed aptamer folding assay by mixing 1uM probe and 1uM 22-bp target together in aptamer folding buffer with fluorogen DFHBI. The fluorescence intensity were detected by CLARIOstar microplate reader at 447/501 Ex/Em. At least 1 aptamer from each subtype yield a statistically significant on/off ratio from using student's t test, except for H1.

![fig1.1](https://static.igem.org/mediawiki/2018/9/94/T--Hong_Kong-CUHK--H1H3H7.png)

*Fig 1.1. Aptamer refolding assay for aptamers of H1, H3, H7. A: aptamer; A+T: aptamer-target RNA pair; +ve: positive control with miniSpinach. 0 aptamer-target pairs for H1, 1 for H3 (purple) and 2 for H7 (blue and orange) had significantly positive on/off ratio. N=3. \*:p<0.05, \*\*:p<0.01, \*\*\*:p<0.005, \*\*\*\*:p<0.0001.*

![fig1.2](https://static.igem.org/mediawiki/2018/8/8a/T--Hong_Kong-CUHK--N1N2N9.png)

*Fig 1.2. Aptamer refolding assay for aptamers of N1, N2, N9. A: aptamer; A+T: aptamer-target RNA pair; +ve: positive control with Spinach. 2 aptamers for N1 (blue and purple), 2 for N9 (blue and orange), and 1 for N2 (orange) had significantly positive on/off ratio. N=3. \*:p<0.05, \*\*:p<0.01, \*\*\*:p<0.005, \*\*\*\*:p<0.0001.*

![fig1.3](https://static.igem.org/mediawiki/2018/b/b1/T--Hong_Kong-CUHK--PB2_Fluoro.jpeg)

*Fig 1.3. Aptamer refolding assay for aptamers of PB2. A: aptamer; A+T: aptamer-target RNA pair; +ve: positive control with Spinach. 2 aptamers for PB2 (blue and green) had significant positive on/off ratio. N=3. \*:p<0.05, \*\*:p<0.01, \*\*\*:p<0.005, \*\*\*\*:p<0.0001.*

### 2. Characterization of Aptamer

#### *1. Specificity*

To test if our probes are specific to their targets, we hybridized the probes with both their supposed targets and other targets. Microplate reader data shows that the aptamer-target pairs are significantly orthogonal.

![fig3.2](https://static.igem.org/mediawiki/2018/8/82/T--Hong_Kong-CUHK--Orthogonality.png)

*Fig.2.1.1 Heatmap showing the microplate reader data from hybridizing different targets and aptamers. This shows that our aptamer-target interaction pairs are orthogonal. N=3.*

![fig3.2](https://static.igem.org/mediawiki/2018/b/bd/T--Hong_Kong-CUHK--Orthogonality_Table.png)

*Fig.2.1.2. Two-Way ANOVA of the data. It shows that signals are changed the most by Aptamer-Target Pairs.*

#### *2. Detection Limit*

For our probe to detect minimal amount of viral RNA, it is necessary to investigate their limit of detection, as defined by the minimum amount of target RNA required to generate signal that is larger than its aptamer only (-ve) signal + 3 standard deviations. It can also be subjectively determined by visibility, though it might be less valid. Minimum amount of target RNA required to obtain a visually distinguishable difference between positive and negative signal was investigated by using 2 model aptamer probes, N2-694 and N9-545, as they represent a less sensitive and a noisier probe respectively. Reaction mixture was set with 2uM probe but in different concentration of target (i.e. 1.5uM, 1uM, 0.5uM, 0.2uM, 0.05uM).

Visually, for N2 probe, more than 0.2uM of target is necessary to visualize the difference, while ~0.2-0.5uM of target is required for N9 probe visualization. (See Fig 2.2.1 and 2.2.2)

![fig2.3.1](https://static.igem.org/mediawiki/2018/f/f4/T--Hong_Kong-CUHK--re_new1.png)

*Fig 2.2.1. Detection limit of N2 probe. 2 uM probe were added to each tube except blank. Blank = buffer + DFHBI.*

*(Up) Bar chart of fluorescence signal by N9 probe-target pairs, using CLARIOstar plate reader with Ex/Em 447/501. Red line indicate the smallest target concentration that form a fluorescence signal significantly larger than the signal arising from aptamer alone by plate reader.*

*(Down) Picture taken by ChemiDoc Imager under SYBR Green mode with Blue Trans Light Excitation.*

![fig2.3.1](https://static.igem.org/mediawiki/2018/0/0a/T--Hong_Kong-CUHK--re_new2.png)

*Fig 2.2.2. Detection limit of N9 probe. 2 uM probe were added to each tube except blank. Blank consisted of only buffer, DFHBI and nuclease free water.*

(Up) Bar chart of fluorescence signal by N9 probe-target pairs, using CLARIOstar plate reader with Ex/Em 447/501. Red line indicate the smallest target concentration that form a signal significantly larger than the signal arising from aptamer alone by plate reader.

*(Down) Picture taken by ChemiDoc Imager under SYBR Green mode with Blue Trans Light Excitation.*

#### *3. Ions Dependency*

Different concentration of sodium, potassium, calcium and magnesium ions were added to the aptamer folding reaction mixture of N2-694 and N9-545 probes, mimicking the addition of nasal fluid to our freeze-dried aptamer kit by household users. These 2 aptamer probes behaved similarly in different selected ions concentration. In the range of the ionic composition of nasal fluid, they performed well for sodium, potassium ions and magnesium ion, though an increase in calcium ions concentration will result in a decrease of fluorescence signal of both probes.

Overall, the aptamer system would not affected by sodium, potassium and magnesium ions in nasal fluid, except calcium ion. Note that the aptamer folding buffer used composed of 10mM Tris-HCl, 100 mM KCl, 5 mM MgCl2.

![fig2.3.1](https://static.igem.org/mediawiki/2018/6/65/T--Hong_Kong-CUHK--Na_salt.png)

*Fig.2.3.1. Sodium ion dependency of N2-694 (left) and N9-545 (right) aptamer probes. Dark blue arrow indicate the approximate sodium ion concentration presented in reaction mixture after the addition of nasal fluid.*

*Both probes showed a good on-off ratio even with the addition of sodium ions.*

![fig2.3.2](https://static.igem.org/mediawiki/2018/6/64/T--Hong_Kong-CUHK--K_salt.png)

*Fig.2.3.2. Potassium ion dependency of N2-694 (left) and N9-545 (right) aptamer probes.*

*Dark purple arrow indicate the approximate potassium ion concentration presented in buffer with the addition of nasal fluid.*

*Both probes demonstrated an increasing signal with the increase in potassium ions concentration. But they performed well when considering the native amount of potassium ions presented in nasal fluid and the buffer.*

![fig2.3.3](https://static.igem.org/mediawiki/2018/4/4e/T--Hong_Kong-CUHK--Ca_salt.png)

*Fig.2.3.3. Calcium ion dependency of N2-694 (left) and N9-545 (right) aptamer probes.*

*Pink arrow indicate the approximate calcium ion concentration presented in buffer with the addition of nasal fluid.*

*Both probes displayed a decreasing signal, with an increase potassium ions concentration. In the range of calcium ionic composition in nasal fluid, though the decreasing signal, both probes still yielded a significant (by 2-Way ANOVA) signals.*

![fig2.3.4](https://static.igem.org/mediawiki/2018/d/d9/T--Hong_Kong-CUHK--Mg_salt.png)

*Fig.2.3.4. Magnesium ion dependency of N2-694 (left) and N9-545 (right) aptamer probes.*

*Green arrow indicate the approximate magnesium ion concentration presented in buffer with the addition of nasal fluid.*

*Both probes displayed a good on/off ratio with the addition of magnesium ion from nasal fluid.*

### 3. Optimization of Aptamer Assay Condition

We were able to monitor the change in fluorescence signal and the time required for signal development using real-time PCR system. N9-694 and N2-545 probes were used.

It was shown that our aptamer-probe pairs generally require only 10 minutes of cooling to give out a detectable signal, which is around 75 degrees Celsius. (Fig 3.1)

![fig3.1](https://static.igem.org/mediawiki/2018/d/d0/T--Hong_Kong-CUHK--re_fig3.png)

*Fig.3.1 Time and Temperature for signal development of N9 and N2 probes. Fluorescence intensity is measured in Bio-Rad Real-Time PCR system. Reaction mixtures are heated at 95 degrees Celsius for 5 minutes, then slowly dropped to 25 degrees Celsius. N=1.*

On the other hand, after refolding is completed, we performed melt curve analysis with the same real-time PCR system. Surprisingly, the melting curve shows that the fluorescence intensity drops quickly at 18-35 degrees Celsius, which does not correspond to the previous data. However, this might be caused by the assymetry of association and dissociation kinetics of DFHBI docking, which was never studied in the previous literature. Another interesting point is that both miniSpinach and aptamer-target pairs share 2 melting temperatures, suggesting a two-step mechanism of DFHBI docking. Nevertheless, the temperature where the probe achieves optimal performance is around 18 degrees Celsius, while room temperature incubation can also achieve a very goof fluorescence signal, which is user-friendly (See Fig 3.2)

![fig3.2](https://static.igem.org/mediawiki/2018/0/03/T--Hong_Kong-CUHK--re_melt.png)

*Fig.3.2 Melting curves of N9 and N2 probes. Fluorescence intensity is measured in Bio-Rad Real-Time PCR system. After refolding, the reaction mixtures are heated from 4 degrees Celsius to 95 degrees Celsius over the course of one hour. N=1.*

### 4. E. coli Expression Test

When we co-expressed the probes and targets in *E. coli* BL21 Star (DE3) provided by [https://2018.igem.org/Team:Hong\_Kong-CUHK/Collaborations NUS Singapore-A team], we did not observe the expected ON/OFF ratio as seen in the in vitro experiments. While we initially hypothesized that this might be due to the low expression level of the probes and the target, as the positive control had low fluorescence comparing to blank as well, we performed total RNA extraction and used the RNA amount that is equivalent to the cell number for in-cell assay. To our surprise, we observed a 7-fold recovery of the fluorescence level of the positive control in total RNA, while none of that was observed in the probe + target pairs. Based on the results, we concluded that while aptamer folding without tRNA scaffold is unfavorable in E. coli, probe target hybridization is inhibited by the terminator following the aptamer. Please note that the design of this assay is following Ong et. al. 2017, showing that the data from the paper might not be accurate. However, due to the lack of time, we did not perform further alterations to improve the design (Fig. 4).

![fig4](https://static.igem.org/mediawiki/2018/b/b9/T--Hong_Kong-CUHK--re_ecoli.png)

*Fig.4 E. coli Probe expression test. (a) Results from initial probe screening using E. coli BL21 (DE3), data retrieved from microplate reader. N=3. (b) Relative fluorescence of total RNA and its comparison to other screening approaches. N=1.*

### 5. Improve a previous biobrick and project – RNA Reporter for Promoter Activity Measurement

We have cloned the lpp promoter-conjugated new RNA reporters into pSB1C3 for assay, including tRNALys3-miniSpinach, tRNALys3-iSpinach-D5 and tRNALys3-dBroccoli. The overnight culture was directly used for DFHBI incubation and plate reader measurement. We found out that all 3 probes are brighter than the original biobrick (Fig.6.1).

![fig5.1](https://static.igem.org/mediawiki/2018/e/ed/T--Hong_Kong-CUHK--re_51.png)

*Fig.5.1 Spinach Aptamer Reporter Test in DH5a. N=1.*

Collaborating with [https://2018.igem.org/Team:Hong\_Kong-CUHK/Collaborations NUS Singapore-A team], they have cloned the same constructs and performed the assay with BL21 Star (DE3), a RNase-E deficient strain. They also performed the assay at different incubation temperatures to see the temperature sensitivity of the RNA reporter. For convenience, only tiSpinach was tested. It is found that tiSpinach is less temperature-sensitive than tSpinach2.1 (Fig.6.2).

![fig5.2](https://static.igem.org/mediawiki/2018/f/f8/T--Hong_Kong-CUHK--re_52.png)

*Fig.5.2 Spinach Aptamer Temperature Sensitivity Test in BL21 Star (DE3). (a) iSpinach is brighter than Spinach2.1 across all temperatures, although only at 45 degrees Celsius is the difference significant. (b) Signal-to-noise ratio calculated from dividing positive signal to untransformed signal. (c) Multiple t test showing that iSpinach is less significantly different for 37 vs 45 comparison than Spinach2.1, showing that it has higher reliaility in reporting heat-induced promoter activity. N=3.*

In conclusion, we have constructed new RNA reporters with better properties than BBa\_K1330000.