iGEM 2019 — Team CSMU_Taiwan

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# Team:CSMU Taiwan/Notebook

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* Team

  + [Team](https://2019.igem.org/Team%3ACSMU_Taiwan/Team)
  + [Attributions](https://2019.igem.org/Team%3ACSMU_Taiwan/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ACSMU_Taiwan/Collaborations)
* Project

  + [Description](https://2019.igem.org/Team%3ACSMU_Taiwan/Description)
  + [Design](https://2019.igem.org/Team%3ACSMU_Taiwan/Design)
  + [Demonstrate](https://2019.igem.org/Team%3ACSMU_Taiwan/Demonstrate)
  + [Medal Criteria](https://2019.igem.org/Team%3ACSMU_Taiwan/Medal_Criteria)
* Lab

  + [Experiments](https://2019.igem.org/Team%3ACSMU_Taiwan/Experiments)
  + [Results](https://2019.igem.org/Team%3ACSMU_Taiwan/Results)
  + [Notebook](https://2019.igem.org/Team%3ACSMU_Taiwan/Notebook)
  + [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol%20)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ACSMU_Taiwan/Parts)
  + [Characterization](https://2019.igem.org/Team%3ACSMU_Taiwan/Characterization)
  + [Improve](https://2019.igem.org/Team%3ACSMU_Taiwan/Improve)
* HP

  + [HP Structure](https://2019.igem.org/Team%3ACSMU_Taiwan/Human_Practices)
  + [Integrated Human Practice](https://2019.igem.org/Team%3ACSMU_Taiwan/Human_Practices#iHP)
  + [Public Engagement](https://2019.igem.org/Team%3ACSMU_Taiwan/Public_Engagement%20)
  + [Entrepreneurship](https://2019.igem.org/Team%3ACSMU_Taiwan/Entrepreneurship%20)
* [Model](https://2019.igem.org/Team%3ACSMU_Taiwan/Model)
* [Safety](https://2019.igem.org/Team%3ACSMU_Taiwan/Safety)

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* Team

  + [Members](https://2019.igem.org/Team%3ACSMU_Taiwan/Team)
  + [Attributions](https://2019.igem.org/Team%3ACSMU_Taiwan/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ACSMU_Taiwan/Collaborations)
* Project

  + [Description](https://2019.igem.org/Team%3ACSMU_Taiwan/Description)
  + [Design](https://2019.igem.org/Team%3ACSMU_Taiwan/Design)
  + [Demonstrate](https://2019.igem.org/Team%3ACSMU_Taiwan/Demonstrate)
  + [Medal Criteria](https://2019.igem.org/Team%3ACSMU_Taiwan/Medal_Criteria)
* Lab

  + [Experiments](https://2019.igem.org/Team%3ACSMU_Taiwan/Experiments)
  + [Results](https://2019.igem.org/Team%3ACSMU_Taiwan/Results)
  + [Notebook](https://2019.igem.org/Team%3ACSMU_Taiwan/Notebook)
  + [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol%20)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ACSMU_Taiwan/Parts)
  + [Characterization](https://2019.igem.org/Team%3ACSMU_Taiwan/Characterization)
  + [Improve](https://2019.igem.org/Team%3ACSMU_Taiwan/Improve)
* HP

  + [HP Structure](https://2019.igem.org/Team%3ACSMU_Taiwan/Human_Practices)
  + [IHP](https://2019.igem.org/Team%3ACSMU_Taiwan/Human_Practices#iHP)
  + [Public Engagement](https://2019.igem.org/Team%3ACSMU_Taiwan/Public_Engagement)+ [Entrepreneurship](https://2019.igem.org/Team%3ACSMU_Taiwan/Entrepreneurship)
* [Model](https://2019.igem.org/Team%3ACSMU_Taiwan/Model)
* [Safety](https://2019.igem.org/Team%3ACSMU_Taiwan/Safety)

![](https://static.igem.org/mediawiki/2019/a/a0/T--CSMU_Taiwan--notebook_.png)

#### Index

* [Protein](#Protein)

  + [March](#Protein_Mar)
  + [April](#Protein_Apr)
  + [May](#Protein_May)
  + [June](#Protein_June)
  + [July](#Protein_July)
  + [August](#Protein_Aug)
  + [September](#Protein_Sep)
  + [October](#Protein_Oct)
* [Selex](#Selex)

  + [May](#Selex_May)
  + [June](#Selex_June)
  + [July](#Selex_July)
  + [August](#Selex_Aug)
  + [September](#Selex_Sep)

### Protein

2019  March

March 7th ~ 28th   NPA & NPB Transformation  ,  Small and Large Scale Production

2019  April

April 1st ~ 9th   NPA & NPB Small and Large Scale Production

April 23rd ~ 27th   NPA & NPB Production

April 25th ~ May 3rd   NPA & NPB Production

2019  May

May 1st ~ 12th   NPA & NPB Production

May 8th ~ 12th   NPA & NPB Production

2019  June

June 28th   NPA new & NPB new Transformation

June 29th ~ July 4th   NPA new & NPB new Small Scale Production

2019  July

July 1st ~ 7th   7/2\_NPA & NPB Production

July 6th ~ 7th   NPA new & NPB new Small Scale Production

July 14th ~ 15th   NPA new & NPB new Small Scale Production test

July 15th ~ 17th   7/16\_NPA & NPB Production

July 22nd ~ 28th   7/23\_NPA & NPB  ,  7/24\_NPA  ,  7/26\_NPA Production

July 29th ~ August 4th   7/30\_NPB  ,  7/31\_NPA-1 & NPA-2  ,  8/1\_NPB-1 & NPB-2

2019  August

August 5th ~ 11st   8/5\_NPB-1 & NPB-2  ,  8/9\_NPA-1 & NPA-2 Production

August 11th ~ 14th   THU protein & 8/12\_NPB Production

August 13th ~ 15th   8/14\_NPA & NPB Production

August 15th ~ 17th   TEV protease sequence in pSB1C3 transformation   &   plasmid miniprep of pET29a

August 15th ~ 20th   HA improved Transformation & HA improved Small Scale Production

August 15th ~ 23rd   8/16\_NPA & NPB Production

August 16th ~ 17th   8/2\_NPB  ,  8/9\_NPA-1 & NPA-2 Protein Dialysis

August 17th ~ 18th   8/12\_NPB  ,  8/8\_NPB-1 & NPA-2 Protein Dialysis

August 17th ~ 21st   TEV PCR  ,  Purification  ,  Digestion

August 20th ~ 27th   8/21\_NPA & NPB Production

August 21st ~ September 2nd   8/22\_HA improved
Production

August 26th ~ 27th   7/24\_NPA Protein Purification

August 26th ~ 30th   8/29\_NPA Production

August 26th ~ September 6th   TEV sequence plasmid miniprep  ,  PCR  ,  Purification and Digestion

2019  September

September 1st ~ 3rd   New & Old NP Production Comparison

September 5th ~ 7th   MIND collaboration

September 8th ~ 17th   Plasmid miniprep of pET29a plasmid  ,  Ligation  ,  Colony PCR

September 11th ~ 17th   9/12\_NPB-1 & NPB-2 Production

September 16th ~ 23rd   Plasmid mini prep  ,  Digestion and Transformation of TEV-pET29a

September 18th ~ 20th   9/19\_NPB Production

September 22nd ~ 25th   9/23\_NPA & NPB Production

September 26th ~ 27th   TEV Small Scale Production

2019  October

October 2nd ~ 6th   10/3\_TEV Production

October 3rd ~ 18th  HA improved & HA original Transformation  ,  Production Comparison

October 15th ~ 18th   10/16\_TEV Production

### Selex

2019  May

   Quantify protein concentration and Test SELEX condition using SELEX tube.

2019  June

SELEX   NPA   first time

2019  July

PCR condition test

2019  August

SELEX   NPA   second time

SELEX   NPB

SELEX   HA3

2019  September

SELEX   HA1

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# Team:CSMU Taiwan/Experiments

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* Team

  + [Team](https://2019.igem.org/Team%3ACSMU_Taiwan/Team)
  + [Attributions](https://2019.igem.org/Team%3ACSMU_Taiwan/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ACSMU_Taiwan/Collaborations)
* Project

  + [Description](https://2019.igem.org/Team%3ACSMU_Taiwan/Description)
  + [Design](https://2019.igem.org/Team%3ACSMU_Taiwan/Design)
  + [Demonstrate](https://2019.igem.org/Team%3ACSMU_Taiwan/Demonstrate)
  + [Medal Criteria](https://2019.igem.org/Team%3ACSMU_Taiwan/Medal_Criteria)
* Lab

  + [Experiments](https://2019.igem.org/Team%3ACSMU_Taiwan/Experiments)
  + [Results](https://2019.igem.org/Team%3ACSMU_Taiwan/Results)
  + [Notebook](https://2019.igem.org/Team%3ACSMU_Taiwan/Notebook)
  + [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol%20)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ACSMU_Taiwan/Parts)
  + [Characterization](https://2019.igem.org/Team%3ACSMU_Taiwan/Characterization)
  + [Improve](https://2019.igem.org/Team%3ACSMU_Taiwan/Improve)
* HP

  + [HP Structure](https://2019.igem.org/Team%3ACSMU_Taiwan/Human_Practices)
  + [Integrated Human Practice](https://2019.igem.org/Team%3ACSMU_Taiwan/Human_Practices#iHP)
  + [Public Engagement](https://2019.igem.org/Team%3ACSMU_Taiwan/Public_Engagement%20)
  + [Entrepreneurship](https://2019.igem.org/Team%3ACSMU_Taiwan/Entrepreneurship%20)
* [Model](https://2019.igem.org/Team%3ACSMU_Taiwan/Model)
* [Safety](https://2019.igem.org/Team%3ACSMU_Taiwan/Safety)

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* Team

  + [Members](https://2019.igem.org/Team%3ACSMU_Taiwan/Team)
  + [Attributions](https://2019.igem.org/Team%3ACSMU_Taiwan/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ACSMU_Taiwan/Collaborations)
* Project

  + [Description](https://2019.igem.org/Team%3ACSMU_Taiwan/Description)
  + [Design](https://2019.igem.org/Team%3ACSMU_Taiwan/Design)
  + [Demonstrate](https://2019.igem.org/Team%3ACSMU_Taiwan/Demonstrate)
  + [Medal Criteria](https://2019.igem.org/Team%3ACSMU_Taiwan/Medal_Criteria)
* Lab

  + [Experiments](https://2019.igem.org/Team%3ACSMU_Taiwan/Experiments)
  + [Results](https://2019.igem.org/Team%3ACSMU_Taiwan/Results)
  + [Notebook](https://2019.igem.org/Team%3ACSMU_Taiwan/Notebook)
  + [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol%20)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ACSMU_Taiwan/Parts)
  + [Characterization](https://2019.igem.org/Team%3ACSMU_Taiwan/Characterization)
  + [Improve](https://2019.igem.org/Team%3ACSMU_Taiwan/Improve)
* HP

  + [HP Structure](https://2019.igem.org/Team%3ACSMU_Taiwan/Human_Practices)
  + [IHP](https://2019.igem.org/Team%3ACSMU_Taiwan/Human_Practices#iHP)
  + [Public Engagement](https://2019.igem.org/Team%3ACSMU_Taiwan/Public_Engagement)+ [Entrepreneurship](https://2019.igem.org/Team%3ACSMU_Taiwan/Entrepreneurship)
* [Model](https://2019.igem.org/Team%3ACSMU_Taiwan/Model)
* [Safety](https://2019.igem.org/Team%3ACSMU_Taiwan/Safety)

![](https://static.igem.org/mediawiki/2019/6/66/T--CSMU_Taiwan--experenment_.png)

#### Index

* [Part I. Protein production](#A)
* [Part II. SELEX](#B)
* [Part III. Analysis of aptamers](#C)

## **Part I. Protein production**

Aptamer, the sequence of DNA, RNA, or peptide that could form a specific functional structure and bind with other molecular, protein, even whole cell, were chosen to be the sensor for distinguishing the type A and B influenza from each other. The production of aptamer need series of selection and examination. In this page, all methods that we ever used in experiment are presented.

During the aptamer selection, large amount of nucleocapsid protein of type A influenza (or NPA) and nucleoprotein of type B influenza (or NPB) were needed as target to select aptamers that can distinguish them from each other (hereafter refer to AptNPA and AptNPB). For the stable supply of NPA and NPB used in SELEX, the target sequences with plasmid were inserted into *E.coli* expression system to produce protein products continuously.

At beginning, target sequence was sent into pET29a plasmid and controlled by lac operon. These plasmids were sent into competent cell BC21(DE3) by heat shock process. After successfully transformed the *E.coli*, these bacteria would be selected by adding antibiotic kanamycin in culture process. Only those bacteria that had pET29a plasmid with target gene would be provided the antibiotic resistance and survived in selection.

The survived *E.coli* would become miniature factory to produce the target protein. The IPTG was added to turn on Lac operon in *E.coli* expression system, thus the recombinant protein would be synthesized and purified by following steps:

1. Induce *E.coli* to produce recombinant protein by adding IPTG in culture process.
2. Break the cell with ultrasonic processor and collect the cell lysate.
3. Centrifuge to separate and collect the suspension from lysate, because our target protein is water soluble.
4. Purify the target protein from lysate.
5. Dialyze the protein to remove imidazole.
6. Finally, get the protein product for SELEX.

To improve the yield of recombinant protein, the optimization of protein production was executed in different periods of experiment. The improvement of method in every period is descripted as below.

#### **Phase 0: Polyhistidine-tag on target protein**

Before starting producing target protein, finding an approach to identifying and separating NPA and NPB from various bacterial proteins is necessary for target protein production. Polyhistidine-tag (or His-tag) is an amino acid motif added at the terminus of target sequence. Expressed His-tagged protein can be detected or separated by the histidine residues on it. This is a cheap and convenience way for researcher to purify the recombinant protein.

The His-tag was added at the C-terminus of the sequence, and the His-tagged protein was purified by Ni-NTA column (see **Fig. 1**). With this method, lots of money could be saved in purification and detection process.

![]( https://static.igem.org/mediawiki/2019/8/85/T--CSMU_Taiwan--experiment_figure1.jpeg)

**Figure 1.**The Ni-NTA column that used in recombinant protein purification.

#### **Phase 1: IPTG condition test**

The IPTG effect in the protein expression was tested during protein production period. The *E.coli* was divided into groups according the time and concentration of IPTG treatment. The bacteria culture curve of all group was measured by spectrophotometer, and protein productivity was determined by western blotting.

#### **Phase 2: Sequence optimization**

The productivity of NPA and NPB was always low even after adjustment of induction condition. Therefore, the sequence of NPA and NPB was suspected to be the reason of low expression of recombinant protein. Website tools such as ATGme and SignalP was applied to optimize sequence of target protein. It can be divided into three section:

1. Removal of rare codons.
2. Detection of restriction site.
3. Prediction the existing probability of signal peptide.

The introduction of these tools work and the reports of optimization are presented in [Model](https://2019.igem.org/Team%3ACSMU_Taiwan/Model#B)
page. After sequence optimization, the new sequences were synthesized and used to restart the protein production process. The productivity of recombinant protein that produced from new sequence was compared with the productivity of product from old sequence. As showed in [Results](https://2019.igem.org/Team%3ACSMU_Taiwan/Results#Optimized%20sequence), the protein productivity is significantly improved in optimized sequence.

#### **Phase 3: New method of dialysis**

At early period of experiment, the dialysis membrane was used to remove imidazole remained in purification process. This method took very long time, and the protein was unstable in high concentration of imidazole for long period of time. As a result, our research team usually wasted much time at dialysis step and got poor product recovery rate.

To solve this problem, new method by ion exchanging column was applied to replace the original method by dialysis membrane (see **Fig. 2**
). Unlike the original dialysis method using concentration gradient to exclude the imidazole out, the new method sorts out the imidazole by centrifuge and preserves the protein on membrane. Thus, it takes less time than before and concentrate the protein product at the same time. This method improves the experiment efficiency dramatically.

![]( https://static.igem.org/mediawiki/2019/2/2a/T--CSMU_Taiwan--experiment_figure2.png)

**Figure 2.**The Ni-NTA column that used in recombinant protein purification.

## **Part II. SELEX**

The aptamers that can detected biomarkers of influenza was selected from random oligonucleotide pool through SELEX. The pool of ssDNA sequences that containing 40 random sites in 87 nucleic acids long was used as probe to react with the NPA, NPB, and the hemagglutinins from H1 and H3 subtype of influenza (hereinafter referred to as HA1 and HA3).

The SELEX brief steps are described below:

1. **Binding:** The ssDNA library is renatured to form tertiary structure in appropriate condition, and react with target protein in binding buffer.
2. **Washing:** Exclude unbound ssDNA with washing buffer.
3. **Elution:** Elute and collect the ssDNA binding on target with elution buffer.
4. **Amplification:** Denature the elution product and amplify by PCR process. The PCR products become library in next round of SELEX.

##### (This SELEX process will repeat 6-8 time to get effective aptamer. )

Just like process protein production, there had arisen bottlenecks with experiment of SELEX. Several examination and adjustments of SELEX method were executed to solve problems in different periods.

![](https://static.igem.org/mediawiki/2019/5/5d/T--CSMU_Taiwan--selex.png)

**Figure 3.** The schematic diagram of SELEX process.

#### **Phase 1. SELEX in column**

In original protocol, the ssDNA probes react with target protein in the special column. As indicated in the reference research, a large amount (0.5~1 mg) of target protein is required for this method. After three months of protein production, 0.9mg of NPA protein was produced to carry out this experiment.

Unfortunately, our produced protein did not be analyzed at that time, thus resulting in the rapid separation between our gel and protein due to the high concentration of sodium solution. It was going to take another long period of time before having enough protein for the next round SELEX gel preparation. Therefore, two alternations were executed to solve this issue: one was to improve our protein yield by optimizing the sequence, another was to seek for other ways to carry out SELEX.

#### **Phase 2. SELEX in microplate**

For the purpose of reducing the usage of protein for SELEX, another approach to execute SELEX in scant target protein was necessary. We found a novel method referred by article on Nucleic Acid Therapeutics. According the research, the aptamer selection was executed in microplate, and requires much less protein than using SELEX tube.

This method was carried out by using NPA as target protein, and the steps is described below:

(All buffer used in steps are the same as described in the [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol) of SELEX. )

#### Phase 2-1

1. **Target Protein solution preparation:** Take 7.5ul of NPA protein(1.22mg/ml), add into 450ml binding buffer(PBS 0.01M).
2. **Coating:** 2 well of the 96-well microplate was coated with 200ul target protein solution per well (4 ug protein/ per well) at 4℃ overnight.
3. **Blocking:** Add 200 ul of 3% BSA to the 2 wells with NPA and other 2 blank wells at 37°C for 2 hr.
4. **Incubation:** The library was denatured at 95℃ for 15 min, cooled immediately in ice for 10 min, and transferred to BSA-blocked blank wells maintained at 37℃ for 40 min. The uncombined ssDNAs were subsequently transferred to the wells coated with NPA at 37℃ for 40 min. (**Fig. 4**)
5. **Washing:** Add 200ul of Washing buffer with Tween 20 for four times.
6. **Elution:** Add Elution buffer and incubate for 90 sec, then transferred to a clean Eppendorf.

##### (The following steps including prescription, PCR and gel electrophoresis are the same as mentioned in SELEX.)

However, nothing appeared in the gel electrophoresis after PCR, we thus suspected that aptamers could have all bound to BSA during the first incubation step. Therefore, several alternations were carried out and be tested as bellow:

#### Phase 2-2

1. **Target Protein solution preparation:**Take 13.5ul of NPA protein (1.22mg/ml), add into 850ml binding buffer(PBS 0.01M).
2. **Coating:** 4 well of the 96-well microplate was coated with 200ul target protein solution (3.8 ug protein/ per well) at 4℃ overnight.
3. **Blocking:** Add 200 ul of 3% BSA to the 4 wells with NPA and other 2 blank wells at 37°C for 2 hr.
4. **Incubation** The library was denatured at 95℃ for 15 min, cooled immediately in ice for 10 min, then transferred to the 2 BSA-blocked blank wells and other 2 blank wells maintained at 37℃ for 40 min. The uncombined ssDNAs were subsequently transferred to the wells coated with NPA at 37℃ for 40 min.( **Fig.4** )
5. **Washing:** Add 200ul of Washing buffer with Tween 20 for twice.
6. **Elution:** Add Elution buffer and incubate for 90 sec, then transferred to a clean 1.5 ml tube.

##### (The following steps that including prescription, PCR and gel electrophoresis are the same as mentioned in SELEX.)

![](https://static.igem.org/mediawiki/2019/2/22/T--CSMU_Taiwan--2-1.png)

**Figure 4.**The schematic diagram that showed the approach to select aptamer in the microplate.

Unfortunately, still nothing appeared in the gel electrophoresis after PCR. There are two possible reasons, one is that aptamers bound to the plates or BSA, which wasn’t quite possible. Another is that aptamers didn’t have sufficient reaction with the coated protein limited to the reaction surface of 96 well microplate.

#### **Phase 3. SELEX in 1.5ml centrifuge tube**

To solve the problem of reaction efficiency, 1.5 ml centrifuge tube was chosen to be the container of SELEX reagent and 3D rotary mixer was used to improve the mixing efficiency.

Finally, the result of DNA concentration in elution product meeting our expectation after series of adjustment of method. Our experiment could be carried on and would not be limited by the protein yield. (the method is presented in [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol)

But the good circumstances didn’t last long, the by-product started appearing in PCR result after 2-3 rounds of SELEX and impacted normal 87nts product yield seriously.

#### **Phase 4. PCR condition optimization**

Because of the by-product effect, all attempting to get a normal aptamer was failed. To find out the reason, all variables that can be control in PCR process were tested. Eventually, we summarize all data and build the PCR model of SELEX. Our finding is showed in the [PCR condition optimization.](https://2019.igem.org/Team%3ACSMU_Taiwan/Model#D)

## **Part III. Analysis of aptamers**

After aptamer selection and sequencing, it was necessary to analyze the binding ability and specificity of aptamer-target binding. ELISA (enzyme-linked immunosorbent assay) was used to test the affinity of our aptamers. The following description is about the method to analyze the properties of aptamer:

#### **Phase 1. Non-specificity ELISA**

Standard ELISA method can be distinguished into two types: specificity ELISA (also called “sandwich” ELISA) and non-specificity ELISA. Because we didn’t have the antibody of any target protein, we chose non-specificity ELISA method to analyze the production of SELEX. ELISA was executed in the steps listed below:

1. Coat the target protein in sequence dilution to the wells of microplate.
2. Aptamers with biotin are added into wells to react with coated protein.
3. Streptavidin with HRP is added to binding the biotin.
4. TMB is added to react with HRP and display blue color.
5. After fixed period of time, add HCl to end the reaction and turn the color of blue into yellow.

##### ( HRP: horseradish peroxidase; TMB: Tetramethyl Benzidine Dihydrochloride )

The different proteins were coated in wells for different test. For example, we could get the affinity of AptNPA with its target by testing the binding ability with NPA, and know the specificity of them by testing the binding ability with NPB.

#### **Phase 2. Competitive ELISA**

After getting result from specificity ELISA, we used competitive ELISA method to get more precise result about the sensitivity of our aptamer. The protein was coated in wells in constant concentration. And added free protein in different concentration as competitive protein. This way could solve the problem that protein can’t fully coat on the surface, and get more accurate date.

#### **Phase 3. Test Strip Experiment**

After testing the titer of our aptamers via ELISA ( see [Result](https://2019.igem.org/Team%3ACSMU_Taiwan/Results#Aptamer%20Selection%20Result) ), the characters of our selected aptamers have been confirmed. The Ap-NPA-4, Ap-NPA-5, Ap-HA3-4, Ap-HA3-6 had the best affinity and specificity among them. Next step, we chose Ap-NPA-4 in the first test scrip tryout.

The Ap-NPA-4 was conjugated to nanogold ( as presented in [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol) ), then two methods were tried out as described below. The detailed device design and concept can be seen at [Design](https://2019.igem.org/Team%3ACSMU_Taiwan/Design).

![](https://static.igem.org/mediawiki/2019/f/f2/T--CSMU_Taiwan--testpaper.gif)

**Figure 5.**The schematic diagram that presented how the test strip works

1. **Direct-Competition:**

   1. Add 8ul of ApNPA-4-nanogold to release pad, and dry it at 37℃
   2. Add 0.25ul of **ApNPA-4/ApNPA-5** on NC paper as test line, and dry it at RT.
   3. Resemble the whole device as shown in **Fig.5**.
   4. Drop 120ul of sample onto sample pad and see its result

2. **Sandwich Competition:**

   1. Add 8ul of ApNPA-4-nanogold to release pad, and dry it at 37℃
   2. Add 0.25ul of NPA(1mg/ml) on NC paper as test line, and dry it at RT.
   3. Resemble the whole device as shown in **Fig.5**.
   4. Drop 120ul of sample onto sample pad and see its result

The result of test strip is presented in [Results](https://2019.igem.org/Team%3ACSMU_Taiwan/Results#Test%20Strip%20Result).

#### **References**

1. Hamula, C. L., Peng, H., Wang, Z., Tyrrell, G. J., Li, X. F., & Le, X. C. (2016). An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes. Methods, 97, 51-57.
2. Aswani Kumar, Y. V. V., Renuka, R. M., Achuth, J., Venkataramana, M., Ushakiranmayi, M., & Sudhakar, P. (2018). Development of hybrid IgG-aptamer sandwich immunoassay platform for aflatoxin B1 detection and its evaluation onto various field samples. Frontiers in pharmacology, 9, 271.
3. Kukol, A., & Hughes, D. J. (2014). Large-scale analysis of influenza A virus nucleoprotein sequence conservation reveals potential drug-target sites. Virology, 454, 40-47.
4. Li, W., Feng, X., Yan, X., Liu, K., & Deng, L. (2016). A DNA Aptamer Against Influenza A Virus: An Effective Inhibitor to the Hemagglutinin–Glycan Interactions. Nucleic acid therapeutics, 26(3), 166-172.
5. Bitaraf, F. S., Rasooli, I., & Gargari, S. M. (2016). DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX. European Journal of Clinical Microbiology & Infectious Diseases, 35(3), 503-510.
6. Tolle, F., Wilke, J., Wengel, J., & Mayer, G. (2014). By-product formation in repetitive PCR amplification of DNA libraries during SELEX. PloS one, 9(12), e114693.
7. Xu, H., Mao, X., Zeng, Q., Wang, S., Kawde, A. N., & Liu, G. (2008). Aptamer-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for protein analysis. Analytical Chemistry, 81(2), 669-675.
8. Cruz-Aguado, J. A., & Penner, G. (2008). Determination of ochratoxin A with a DNA aptamer. Journal of agricultural and food chemistry, 56(22), 10456-10461.
9. Duan, Y., Gao, Z., Wang, L., Wang, H., Zhang, H., & Li, H. (2016). Selection and identification of chloramphenicol-specific DNA aptamers by Mag-SELEX. Applied biochemistry and biotechnology, 180(8), 1644-1656.
10. González, V. M., Martín, M. E., Fernández, G., & García-Sacristán, A. (2016). Use of aptamers as diagnostics tools and antiviral agents for human viruses. Pharmaceuticals, 9(4), 78.

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# Team:CSMU Taiwan/Results

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* [Model](https://2019.igem.org/Team%3ACSMU_Taiwan/Model)
* [Safety](https://2019.igem.org/Team%3ACSMU_Taiwan/Safety)

![](https://static.igem.org/mediawiki/2019/6/69/T--CSMU_Taiwan--result_.png)

#### Index

* [Expression Result](#Expression)
* [1. Expression](#Expression Result)
* [2. Purification](#Purification)
* [3. Dialysis &Concentration](#Dialysis)
* [4. Optimized sequence](#Optimized sequence)
* [Aptamer Selection Result](#Aptamer Selection Result)
* [1. Evolution of SELEX protocol](#Part 1. Evolution of SELEX protocol)
* [2. SELEX result](#Part 2. SELEX result)
* [3. Qualitative analysis](#QualitativeAnalysis)
* [4. Future work](#Part 4. Future work)
* [Test Strip Result](#Test Strip Result)

## Definition for Abbreviations

### Abbreviation of protein:

* **NPA**: Influenza A virus (A/Michigan/297/2017(H1N1)) nucleocapsid protein (NP) gene, complete cds
* **NPB**: Nucleoprotein for Influenza B virus (B/Colorado/16/2017) nucleoprotein (NP) gene, complete cds
* **HA1**: hemagglutinin of Influenza A H1N1 (A/New York/18/2009)
* **HA3**: hemagglutinin of influenza A H3N2 (A/Perth/16/2009)

### Abbreviation of aptamers:

* **Apt**: aptamer
* **Apt- NPA**: aptamer which binds to NPA
* **Apt- NPB**: aptamer which can bind to NP
* **Apt-HA1**: aptamer which can bind to HA1
* **Apt-HA3**: aptamer which can bind to HA3

### Abbreviation of the SELEX experiment result:

* **Ft**: fluid collected in the tube after the incubation of SELEX
* **W“i” (or w“i”)**: fluid collected after mixing with washing solution. “i” indicates the number of washing round
* **E“i” (or e“i”)**: fluid collected after mixing with elution buffer. “i” indicates the number of eluting round
* **NPA-“i”**: the serial number “i” of the Apt-NPA of all the clones we obtained
* **NPB-“i”**: the serial number “i” of the Apt-NPB of all the clones we obtained
* **HA1-“i”**: the serial number “i” of the Apt-HA1 of all the clones we obtained
* **HA3-“i”**: the serial number “i” of the Apt-HA3 of all the clones we obtained

## Expression Result

As mentioned in the Experiment section, the production of protein can be roughly divided into three parts: The expression of E.coli, the purification of expressed proteins, and the dialysis and concentration of purified protein.

### Part 1. Expression

After expressing our target protein( [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol#overexpression)), the products were centrifuged to separate the solubles and precipitates. The samples were then checked by SDS page.(Figure 1)

Most of the production are found in S and S1, indicating that the expressed proteins are highly soluble. The dark band also indicates that both of them are the main product of E.coli expression.

**Estimated Protein Molecular weight:**

**NPA:**Sequence length 1494bp→498 codon +6 Histidine

             110Da average/per amino acid→55440Da=**55.4kDa**

**NPB:** Sequence length 1680bp→560 codon +6 Histidine

            110Da average/per amino acid→ 62260Da=**62.3kDa**

![](https://static.igem.org/mediawiki/2019/8/8d/T--CSMU_Taiwan--result1.png)

* 1-a :NPA expression, the framed band is our target NPA protein, approximately 55kDa.
* 1-b :NPB expression, the framed band is our target NPA protein, approximately 65kDa.

Most of the production for both the NPA and NPB are in the soluble solution, indicating that the proteins are soluble.

As seen in Figure 1, many other non-target protein exists in the solution. Hence, purification is taken out in the next step.

### Part 2. Purification

We purified our target protein using S1 from the previous step. ( [Protocol](https://2019.igem.org/Team%3ACSMU_Taiwan/Protocol))The samples were then checked by SDS page and Western Blot. From SDS page, we are able to confirm if we removed other proteins and purified our protein thoroughly.(Figure 2)

From the SDS page, we can see that most of the target protein are found in Elution buffer, whereas the other proteins has been washed down during the purification steps, left in L, FT, and W.

![](https://static.igem.org/mediawiki/2019/2/20/T--CSMU_Taiwan--result2.png)

* 2-a: An obvious band appears from E1~E8 as framed at 55kDa, the estimated molecular weight of NPA. Barely any other band appear in the elution solutions, indicating that most non-target proteins has been washed down.
* 2-b: An obvious band appears from E1~E5 as framed at 65kDa, the estimated molecular weight of NPB. Barely any other band appear in the elution solutions, indicating that most non-target proteins has been washed down.

Therefore, we confirmed that we purified our target protein thoroughly.
Still, we performed Western-Blot to double check whether the product on the estimated sites were our target protein or not.

We added six His-tag to our target protein. Hence, from western blot, we are able to confirm if the proteins we purified are our target proteins by using antibodies who recognize His-tag. The primary antibodies bind to protein with His-tags, secondary antibodies then binds to primary antibodies, and can be recognized by Fluorescent dye. (Figure 3)

From Western-Blot, we can see that the bands were the same sites shown in the SDS-page.(Figure 2) This proves that the purified protein in the elution sites contains His-tag.

![](https://static.igem.org/mediawiki/2019/1/15/T--CSMU_Taiwan--result3.png)

* 3-a: An obvious band appears from E1~E8 as framed at 55kDa. Indicating that the proteins on the correct site are our target NPA protein.
* 3-b: An obvious band appears from E1~E5 as framed at 65kDa. Indicating that the proteins on the correct site are our target NPB protein.

We have confirmed that the main products in the eluting solution are our target protein.

### Part 3. Dialysis &Concentration

The high concentration of imidazole contained in the protein after purification could cause protein self-degeneration or aggregation, effecting the incubation between protein and Carboxyl gel in the next step. Moreover, the concentration was too low to be efficiently used. Thus, we dialyzed and concentrated the purified protein via Dialysis tube.

![]( https://static.igem.org/mediawiki/2019/f/f7/T--CSMU_Taiwan--result4.png)

Comparing Fig.4-a and Fig.4-b, the concentration has become higher for more efficient use.

This proved that our method was successful.

### Part 4. Optimized sequence

Since the protein yield was low and wasn’t able to supply the need for SELEX , we applied some online tools to optimize the sequence.For example, ATGme to replace the rare codon into the ones that E.coli use , in addition, to examine unexpected restriction sites. After this program, the protein yield became larger as seen in Figure 5 and Figure 6.

![](https://static.igem.org/mediawiki/2019/e/e5/T--CSMU_Taiwan--result5.png)

![](https://static.igem.org/mediawiki/2019/3/3d/T--CSMU_Taiwan--result6-1.png)

Comparing Fig.6-a with Fig.6-b, it is obvious that the yield of protein increased tremendously.

## Aptamer Selection Result

### Part 1. Evolution of SELEX protocol

#### **Phase 1: SELEX tube**

As indicated in the reference papers, a large amount(0.5~1 mg ) of target is required for this method. After three months of protein production, we finally had 0.9 mg of NPA protein to carry out this experiment.

Unfortunately, we did not dialyze our produced protein at that time, resulting in the rapid separation between our gel and protein due to the high concentration of sodium solution. It was going to take another long period of time before having enough protein for a next round SELEX gel preparation. Therefore, we made two alternations to solve this issue:

1. Improve our protein yield by adjusting the sequence[(see Modeling)](https://2019.igem.org/Team%3ACSMU_Taiwan/Model#B)
2. Seek for other ways to take out SELEX.

However, we still collected useful data regarding to the best environment for aptamer refolding in this experiment.

The amount of the by-products :

When aptamers refold in the ice , there were less by-products than when they refold in room temperature.(Figure 7) Thus, we kept the aptamers on ice after degeneration in the following experiments

![](https://static.igem.org/mediawiki/2019/1/13/T--CSMU_Taiwan--resultfigure6.png)

#### **Phase 2: 96 well plate**

In the purpose of reducing the usage of protein for SELEX, we looked over papers and found some papers that perform SELEX in 96- well plate required much less protein.

However, the result for this method did not work well in our experiment(Figure 8). We tried two methods as described in the [Experiment](https://2019.igem.org/Team%3ACSMU_Taiwan/Experiments) section, but nothing appeared in the gel electrophoresis after PCR.

![](https://static.igem.org/mediawiki/2019/5/57/T--CSMU_Taiwan--resultfigure7.png)

In conclusion, we assumed that insufficient mixing between the aptamer pool and coated protein limited to 96-well plate could lead to separation of all aptamers before Washing.

#### **Phase 3: SELEX in Eppendorf**

We concluded that:

1. A smaller operation space for SELEX can reduce the usage of protein.

2. Sufficient mixure between aptamer pool and target protein is required

3. It is more predictable and easy to control/track when protein are incubated with the Carboxyl Gel.

Thus we used Eppendorf as our SELEX tube, and successfully got the following results mentioned in Part 2.

### Part 2. SELEX result

#### A. SELEX

1. The protocol of SELEX(protocol) involves three solutions: flowthrough, washing, and elution, which respectively include aptamers that didn’t bind to anything, aptamers that bound to the Gel but not the target protein, and aptamers that bound to the target protein.
2. **Unspecific Test:**

   To ensure that all aptamers we acquired from the elution bound to target protein instead of the Carboxyl Gel, we performed unspecific binding test. The protocol was identical to the normal SELEX, just that no protein were incubated on the Gel.(Figure 9)
![](https://static.igem.org/mediawiki/2019/e/ec/T--CSMU_Taiwan--resultfigure8.png)

We concluded that aptamers that bound to Carboxyl Gel were all washed down during the washing steps.

3. In our experiment, Two peeks appear after each round of SELEX, respectively after the washing and eluting step, which indicates the complete separation between the aptamers that didn’t bind to the protein incubated on the gel(washing) or the tightly bound aptamers (elution). (Figure 10).
![](https://static.igem.org/mediawiki/2019/5/55/T--CSMU_Taiwan--resultfigure9.png)

After practicing several rounds(5~6) of SELEX, we chose the eluting peeks as our selected aptamer pools, which were expected to have high specificity towards our target protein, and were then sent to be sequenced.

4. The following are the aptamer pools we chose to clone in the next step
![](https://static.igem.org/mediawiki/2019/d/dc/T--CSMU_Taiwan--resultfigure10.png)

Fig 11. Gel electrophoresis result after SELEX round 6 of NPA

![](https://static.igem.org/mediawiki/2019/3/3b/T--CSMU_Taiwan--resultfigure11.png)

Fig 12. Gel electrophoresis result after SELEX round 6 of NPB

![](https://static.igem.org/mediawiki/2019/1/11/T--CSMU_Taiwan--resultfigure12.png)

Fig 13. Gel electrophoresis result after SELEX round 6 of HA1

![](https://static.igem.org/mediawiki/2019/c/c3/T--CSMU_Taiwan--resultfigure13.png)

Fig 14. Gel electrophoresis result after SELEX round 6 of HA3

### Part 3. Qualitative analysis

#### **A. TA cloning**

1. Aptamer pools were cloned into pGEM-T vectors with TA cloning kit, and were amplified and ligated with pGEM-T vector transformed into E.coli DH5α cells. Clones obtained were screened with M13 forward and reverse primers.
2. The ones that were at the length of 268np (181+87) indicates that the sequence has been successfully transformed into E.coli (Figure 15), and these positive colonies were then sent to be sequenced.
![](https://static.igem.org/mediawiki/2019/d/d4/T--CSMU_Taiwan--resultfigure14.png)

#### **B. Sequence analyzation**

1. Sequence were analyzed via NCBI Basic Local Alignment Search Tool, BLAST.
   * NPA[PDF](https://static.igem.org/mediawiki/2019/d/d6/T--CSMU_Taiwan--resultpdf1.pdf)
   * NPB [PDF](https://static.igem.org/mediawiki/2019/d/d1/T--CSMU_Taiwan--resultpdf2.pdf)
   * HA1 [PDF](https://static.igem.org/mediawiki/2019/7/73/T--CSMU_Taiwan--resultpdf3.pdf)
   * HA3 [PDF](https://static.igem.org/mediawiki/2019/6/62/T--CSMU_Taiwan--resultpdf4.pdf)

#### **C. ELISA-Characterization of selected aptamers**

1. The titer value of selected aptamers were determined by ELISA.
2. Pre-progress:
   Amplify the sequenced DNA by PCR, using primers labelled with Biotin to product aptamers with biotin marker.
3. Two types of ELISA were practiced: Non-competitive ELISA and Competitive ELISA.
4. Purpose:

   To confirm the titer value of aptamer towards target protein. According to the coated protein, we are able to test two important character we intend to understand about the selected aptamers:

* The Affinity→ by coating target protein
* The Specificity→by coating un-target protein.

#### Non-competitive ELISA:

1. **Method:**

   Create a series of protein with different concentration via serial dilution. Coat these protein respectively to 96-well microplate. After blocking, add primary antibodies, the selected aptamers in this experiment. The following steps are the same as described in the Protocol.
2. **Expectation:**

   If the affinity was high enough, as the concentration of target protein rises, the bound aptamer would increase too, resulting in the darker color presented.
3. **Result:**

   We found out that the result for NPA, NPB and HA3 were more significant.
   Following are their data:

![](https://static.igem.org/mediawiki/2019/0/0c/T--CSMU_Taiwan--resultfigure15.png)

Apt-NPA-5 had higher affinity toward NPA protein and lower affinity toward NPB, which indicates its high specificity . As a result ,we chose them as our sample in the next test scrip part.

![](https://static.igem.org/mediawiki/2019/1/10/T--CSMU_Taiwan--resultfigure16.png)
![](https://static.igem.org/mediawiki/2019/0/02/T--CSMU_Taiwan--resultfigure17-1.png)
![](https://static.igem.org/mediawiki/2019/f/f0/T--CSMU_Taiwan--resultfigure18.png)
![](https://static.igem.org/mediawiki/2019/c/cb/T--CSMU_Taiwan--resultfigure19.png)

#### Competitive ELISA:

1. **Method:**

   Coat the protein to 96-well microplate, each well containing the same amount of proteins. After blocking, add primary antibodies, the selected aptamers, at the same time, add protein in different concentration as competition. The following steps are the same as described in the Protocol.
2. **Expectation:**

   If the affinity was high enough, as the concentration of competition protein rises, the amount of bound aptamers would decrease, resulting in the lighter color presented.
3. **Result**

   We found out that NPA had the most significant result. The followings are its data:
   NPA(Figure 21)

   ![](https://static.igem.org/mediawiki/2019/e/e1/T--CSMU_Taiwan--resultfigure20.png)

**Conclusion:**We concluded that NPA-4, NPA-6 had outstanding affinity and specificity towards its target protein. Therefore, we chose them as the sample in the next part.

### Part 4. Future work

Other aptamers failed to perform good affinity or specificity. This could be explained by the biotin labeled mechanism: Since only one of the strands of amplified aptamers was labelled with biotin during PCR, only 50% of biotin was able to bind to the right strand that had the function to distinguish the target protein. To solve this problem, we are going to use forward primer labeled with biotin to acquire data in the future.

## Test Strip Result

As mentioned in the [Experiment](https://2019.igem.org/Team%3ACSMU_Taiwan/Experiments) ,we tried two different methods:

![](https://static.igem.org/mediawiki/2019/3/37/T--CSMU_Taiwan--result6.png)

1. **Direct-Competition:**
   1. Method:
      0.25ul of Apt-NPA-4and Apt-NPA-5were coated on the NC paper as test lines. Then 120ul of samples containing 400X diluted 0.84mg/ml NPA protein were loaded onto sample pad.
   2. Expectation:
      One site of NPA in the loaded samples should bind to Apt-NPA-4-Nanogold when flowing through release pad, while another site of NPA should bind to Apt-NPA-5 coated on the NC paper. Therefore, the test line should present the strawberry color of Nanogold particle.
   3. Result:
      As seen in Figure 22, no color was shown on the test line.
   4. Future prospects:
      There are two possible reasons why no color was presented: one is that only one of the two strains aptamers bound to NPA, another is that concentration on the NC paper was too low. We intend to try these conditions in the near future.
2. **Sandwich Competition:**
   1. Method:
      0.25ul of NPA(1.94mg/ml)was coated on the NC paper as test line. Then 120ul of samples containing 400X diluted 0.84mg/ml NPA protein were loaded onto sample pad.
   2. Expectation:
      NPA in the sample should bind to Apt-NPA-4-Nanogold when flowing through release pad,. Therefore, the higher the concentration of NPA was in the sample, the lighter the color of test line would be.
   3. Result:
      As seen in Figure 22 ,a light circle of strawberry red was shown on the test line.

      We then tried other conditions including:

      * **Different concentration of NPA on the NC paper**
        Purpose& Method:
        Because the test line color was too light in the previous experiment, we tried to increase the concentration of NPA on NC paper by dropping more NPA. In other words, we added more drops, while every drop remains 0.25ul .

        Result:

        ![](https://static.igem.org/mediawiki/2019/6/6c/T--CSMU_Taiwan--result7.png)

        The color of test line became darker as we add 1 drop, 2 drops and 3 drops. The color remains almost the same after adding 3 drops.(Figure 23)
        **Conclusion:**
        Adding 3 drops of 0.25ul NPA(1.94mg/ml) was the most suitable condition in our experiment.
      * **Different concentrations of NPA in the sample**

        Purpose& Method:
        We applieddifferent dilution fold to NPA in our sample to look for the threshold concentration, the concentration that competes all the aptamers away, resulting in the fade color of test line.

        Result:
        The color turned lighter when the concentration of NPA rises. The darkest was PBS, then 800 fold diluted, and the lightest was 100 fold diluted. However, color still exists under this condition.(Figure 24)

        ![](https://static.igem.org/mediawiki/2019/9/91/T--CSMU_Taiwan--result8.png)

        Conclusion: This double-proved our experiment was successful. Color goes lighter when the concentration of target protein rises
      * **Different concentration of Apt-NPA-4 conjugated with Nanogold particles.**

      Purpose&Method:
      We suspected that the amount of aptamers conjugated to Nanogold particle was not enough, so that the result between coated NPA and sample NPA wasn’t as significant as we expected to be.We then respectively conjugated 1ug, 2.5, 5ug to Nanogold particle.

      Result:

      ![](https://static.igem.org/mediawiki/2019/f/f8/T--CSMU_Taiwan--result9.png)

      ![](https://static.igem.org/mediawiki/2019/a/ae/T--CSMU_Taiwan--result10.png)

      ![](https://static.igem.org/mediawiki/2019/a/a0/T--CSMU_Taiwan--result11.png)

      As seen in Figure 25, no color is shown when 1ug of Apt-NPA-4 is conjugated to Nanogold. Although no color is shown under naked eyes when 2.5 ug or 5ug of Apt-NPA-4 is conjugated to Nanogold particles, after adjusting the contrast value, the color of test line shows up. Colors can be seen when loading PBS of both 2.5ug and 5ug and 400 fold dilution of NPA at 5ug. No color can be seen when loading 400 folds diluted NPA at 2.5ug.(Figure 26, Figure 27)

      Conclusion:
      The best condition we acquired now is:
      Add 3 drops(0.25ul/drop) of NPA(1.94mg/ul) on NC paper; Conjugate 2.5ug of Apt-NPA-4 with Nanogold particles.
      The threshold is 400 folds diluted NPA(0.84mg/ml).

- **Full Wavelength Scan**

  Purpose& Method:
  To confirm that Apt-NPA-4 has successfully conjugated to Nanogold particle, we tested Apt-NPA-4-Nanogold under Full Wavelength Scan. Blank using Nanogold particles to remove background of Nanogold absorbance.

  Expectation:
  Aptamers are ssDNAs, therefore if aptamers were conjugated to Nanogold, a peak would be anticipated to show up at 260nm.

  Conclusion:
  1ug, 2.5ug and 5ugof Apt-NPA-4 conjugated to Nanogold all showed a peak at 260nm. Indicating that three of them are all successfully conjugated.

  ![](https://static.igem.org/mediawiki/2019/7/78/T--CSMU_Taiwan--result12.png)