Difference between revisions of "Team:CSMU Taiwan/Experiments"

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<p>We coated different protein in well for different test. For example, we could get the titer of AptNPA by binding ability with NPA, and know its specificity by binding ability with NPB.</p>
 
<p>We coated different protein in well for different test. For example, we could get the titer of AptNPA by binding ability with NPA, and know its specificity by binding ability with NPB.</p>

Revision as of 06:16, 20 October 2019

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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.

Part I. Protein production

　　　　　　　　　　　　　　　　　

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(DE 3) 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 resistence and survived in selection.

The survived E.coli would become useful miniature factory to produce the target protein. And we induced them to express our target protein by following steps:

  1. Induce E.coli to produce recombinant protein by adding IPTG in culture process.
  2. 2Break 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, we get the protein product for SELEX.


To improve our protein yield, we keep optimizing the process of protein production. Thus, our protein production method went through several period of change as following description:



Phase 0: Adding His tag into sequence

Before starting producing target protein, it was needed to find a way for identifying and separating our protein from various proteins. The His tag on the plasmid would be added at the C-terminus of the target proteins in protein expression. With that, we could easily purify our protein product with Ni-NTA column and marked the recombinant protein in western blotting process.



Phase 1: IPTG induction

After transformation of E-coli, we can start to multiply these mini “protein factory” and use IPTG to induce them generate protein products. We try to build model of IPTG induction and find out the best condition. You can view our result at IPTG condition test.



Phase 2: Sequence optimization

Because the productivity of NPB is always low and not enough for SELEX use. We decide to optimize the sequence of NPA and NPB, and use the optimized sequence to restart the protein production. We use online tools such as ATGme and SignalP to remove rare codon and restriction site, and exam the existing probability of signal peptide. You can see how it works at sequence optimization. After that, we test the new sequence function and compare with the old sequence, as a result, the protein productivity is significantly improve in sequence optimization sequence optimizationAfter that, we test the new sequence function and compare with the old sequenceas a result, the protein productivity is significantly improve in optimized sequence system.



Phase 3: New method of dialysis

At first, we use dialysis membrane to remove imidazole remained in purification. This method takes very long time, and the protein is unstable in high concentration of imidazole. For that, we often waste much time on dialysis and get a bad product recovery rate. To solve this problem, we use ion exchange column to replace the dialysis membrane. 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 our experiment efficiency dramatically.

Part II. SELEX

Our team choose aptamer as tool to distinguish the target protein from each other. The specific aptamer is selected from random oligonucleotide pool through SELEX. We used ssDNA of 87 nucleotides long that contain 40 random sites as probe to select aptamers against nucleoprotein and nucleocapsid protein from influenza. The SELEX steps is 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 protein production, we modify the protocol of SELEX several times to ensure our experiment can continue and get aptamers we want.



Phase 1.SELEX in column

At start, we referred protocol from research paper in recent years. The ssDNA probes react with target protein in the special column. As indicated in the reference papers, a large amount (0.5~1 mg) of target protein is required for this method. After three months of protein production, we finally had 0.9mg of NPA protein to carry out this experiment.



Unfortunately, we didn’t dialyze our produced protein 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, we made two alternations 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

In the purpose of reducing the usage of protein for SELEX, we searched over papers and found some that performed SELEX in microplate requires much less protein than using SELEX tube. We carried out the method using NPA as target protein as described below. All solutions are the same as described in the 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. (figure 2-1)
  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, we made some slight alternations:

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.(figure2-1)
  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 EppendorfTake 13.5ul of NPA protein (1.22mg.ml), added into 570ml binding buffer (PBS 0.01M).

The following steps including prescription, PCR and Gel electrophoresis are the same as mentioned in SELEX.

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.



SELEX in 1.5ml centrifuge tube

To solve the problem of reaction efficiency, we tried to use 1.5 ml centrifuge tube as device and mixed reactant with 3D rotary mixer. As the result, we successfully got ideal result of elution product, and formulated a new method of SELEX in Eppendorf. (see 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, we fail in all attempt to get a normal aptamer. But we never give up and keep trying test every variable we can control in PCR process. As a Chinese proverb goes: “Heaven helps those who help themselves,” we summarize all data and build the PCR model in SELEX. You can see our finding in the PCR condition optimization.

Part III. ELISA

After aptamer selection and sequencing, we needed to analysis the binding ability and specificity of aptamer-target binding. We used ELISA (enzyme-linked immunosorbent assay) as method to test the titer of our aptamers. The following description is about the method we applied.



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 buy the antibody of our target and our protein had been purified, we chose non-specificity ELISA method as following steps:

  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



We coated different protein in well for different test. For example, we could get the titer of AptNPA by binding ability with NPA, and know its specificity by 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.