iGEM 2022 — Team nu-kazakhstan

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Notebook

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Describe all the safety issues of your project.{% endblock %}
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# Wetlab

## Cycle 1: Cloning (Engineering of E.coli BL21)

![](https://static.igem.wiki/teams/4389/wiki/engineering-success/engineering-success/1.png)

### Design

**Primer design of 1st and 4 sushi**

---

The B5R protein is generally produced in eukaryotic cells in E.coli. Therefore, apart from synthesizing the full sequence, we planned to
synthesize the first sushi ectodomain of B5R. Since we need a proper
expression system for aptamer and antibody testing, pET23a was suggested
as a satisfactory plasmid vector. So, we made three primers:- BBa\_K4389008
- BBa\_K4389009
- BBa\_K4389010

![](https://static.igem.wiki/teams/4389/wiki/engineering-success/engineering-success/promoter.png)

### Build

**PCR amplification, Preparation of competent DH5a cells, Restriction,
Ligation, PCR purification, Transformation, Plasmid extraction, Colony PCR,
Gel extraction**

---

Inserts from pVAX and pET23a vector were firstly restricted and purified.
Then it was transformed into BL-21 E. coli after ligation. Colony PCR and sequencing were done to select the
DNAs with successful assembly.

### Test

**DNA sequencing on Genetic DNA Analyzer**

---

The results of PCR colony and sequencing demonstrated proper ligation of
vector and insert.

![](https://static.igem.wiki/teams/4389/wiki/engineering-success/engineering-success/cycle-2.png)

Figure 3. Three possible reasons for failed experiment

### Learn

**Troubleshooting from benchling**

---

For this cycle, we learned that we have successfully assembled the
construct. Moreover, after brainstorming problems from our lab, we learned
that:

1. We need to add a small amount of water (30ul) instead of elution buffer
2. After analyzing different protocols, we decided to incubate ligation
   products for 20min
3. The suggested volume in protocols for SYBR safe was high which made
   bands invisible under UV; thus, we decided to add only 5ul of it

## Cycle 2: Protein expression

![](https://static.igem.wiki/teams/4389/wiki/engineering-success/engineering-success/2.png)

### Design

**144 different conditions for control**

---

The preliminary data from our superuser group demonstrated that the
protein expression was not observed in SDS-PAGE electrophoresis. Thus, we
decided to check the expression of B5R protein in BL21(DE3) E. coli under different conditions. The conditions varied depending on time
(2h,4h,6h,16h); temperature (19°C, 37°C, 37°C); IPTG concentration (0mM, 0.2mM, 0.5mM, 1mM).

### Build

**Samples were prepared for test expression with SDS-PAGE**

---

We run SDS-PAGE and Western Blot electrophoresis in BioRad protein
electrophoresis.

### Test

**Protein B5R was not expressed**

---

To test electrophoresis gels, we searched for significantly large bands
around 6 and 25 kDa for 1st and 4 sushi B5R proteins, respectively. Also,
the bands were analysed in Western blot for proper protein folding.

### Learn

**Reasons why:**

---

We did not find any large observable bands in gels. The present small
bands around the 6 and 25 kDa area were not supported by Western blot.
This means that there was no or minimal expression of our protein. The
possible reasons can be:

1. Toxicity of B5R protein to BL-21 E. coli which kills them without allowing for observable protein expression.
   Thus, we suggested inserting the signal sequence for Sec (secretion)
   system into our B5R sequence.
2. The failed expression system in our pET23a plasmid. It can be possible
   that our sample of initial vectors may have a defective T7 expression
   system.
3. Another issue may result from non-optimized B5R codon sequences for
   BL21 E. coli.

Thus, we suggested using a different plasmid pET22b plasmid with an
already inserted signal sequence. This way we can tackle the two
above-mentioned problems. Also, we planned to order codon-optimized B5R
sequences. However, we did not have enough time to finish these new
suggestions until the deadline for the wiki freeze. Therefore, we focused
mainly on three other antigens (L1, A27, A33) to test our optic fiber
biosensors. Since aptamers can be used instead of antibodies, we planned
to functionalize them on antibodies as well.

# Drylab

## Cycle 3.1: Optic fibre functionalization with L1, A27, and A33

![](https://static.igem.wiki/teams/4389/wiki/engineering-success/engineering-success/3.png)

### Design

**Designing optic fibers**

---

Optical fibres are an innovative way to make biosensors to detect
specific molecules in different media. They are cheap, sustainable,
extremely safe and experiments have shown high sensitivity of detection -
up to attomolar 10-18 concentrations of molecules. Ordinary
optic fibres can be turned into biosensors by attaching antibodies of the
target molecules onto them in a process called
‘functionalization’, then immersed into solutions with varying
concentrations of the target proteins, and their responses can be
analysed

### Build

**Functionalizing optical fibres with antibodies of proteins of Vaccinia virus**

---

Since protein B5R was not expressed in our engineered E.coli, the optical fibres were functionalized with antibodies of other
proteins of Vaccinia virus: L1, A27 and A33.

### Test

**Detecting Vaccinia virus proteins using optical fibre biosensors**

---

We conducted tests with two types of optic fibre biosensors: biosensors
functionalized with single antibodies and multiple antibodies (multiplex
biosensors). Biosensors with single antibodies have been tested for
detection of their antigen in PBS solution, while the multiplex sensors
were tested both in PBS and sewage water. The results were analysed using
the software our team has devised (see [here](https://2022.igem.wiki/nu-kazakhstan/software)).

![](https://static.igem.wiki/teams/4389/wiki/engineering-success/engineering-success/cycle-3.png)

### Learn

**Optical fiber biosensors detect proteins and changes in their concentrations
in PBS and sewage water**

---

Overall, the optical fibre ball resonators functionalized with single
antibodies and the multiplex biosensors succeeded in detecting the changes
of concentrations of the target antigens in PBS with high sensitivity.
Furthermore, the multiplex sensors were observed to successfully detect
antigen’s concentration changes in samples of synthetic sewage
water, indicating that it was functional at conditions close to the real
environment.

## Cycle 3.2: Aptamer designing

![](https://static.igem.wiki/teams/4389/wiki/engineering-success/engineering-success/4.png)

### Design

**Using aptamers instead of antibodies**

---

Aptamers are nucleotide sequences that specifically bind to target
molecules, and have advantages of being cheap and accessible over
antibodies, and thus it is reasonable to functionalize optical fibres with
aptamers and test them on detecting the target molecules. The conventional
way to make aptamers is via a laborious process called SELEX that usually
takes a long time, and there is open-source software MAWS made by iGEM
Heidelberg 2015 team that we can use to make aptamer sequences for
vaccinia virus proteins.

### Build

**Using MAWS to make aptamer sequences**

---

We used the software MAWS to make DNA aptamer sequences for a Vaccinia
virus protein L1 (as we had limited time, for one protein only).

### Test

**Conducting in-silico tests on aptamers**

---

Then we made the 3D structures of the DNA aptamers and simulated a
docking model with aptamers and their target molecules and analysed
possible interactions they might have.

### Learn

**Aptamers are ordered for in-vivo tests**

---

The results show low binding energy, high confidence scores, high rmsd
values and seemingly good amount of hydrogen bonds in the binding of
aptamers and the protein. However, the results are rather ambiguous, and
it is hard to say if the aptamers and proteins form a good complex with
high confidence, therefore, for reliable validation of the aptamers, we
ordered them to conduct further in-vivo tests on their binding.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments

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## Summary

We developed a fiber-optic-based ultra-sensitive biosensor for the rapid detection of
three OPXVs proteins in the environment. Our results demonstrated the
following:

* The insertion of B5 (1st sushi and all 4 sushi ectodomains) into the
  pET23a plasmid
* No observed synthesis of B5 protein
* Successful expression of L1, A27, A33 proteins
* Functionalization of anti-L1, anti-A27, Anti-33 antibodies on optic
  fiber
* Detection of L1, A27 and A33 antigens by our optic-fiber biosensor in
  PBS and sewage water.

## Molecular cloning

#### Amplification

Figure 1 shows the picture of agarose gel of PCR products encoding 1st and 4
sushi domains of B5R protein. The forward primer was the same for
sequences encoding both 1st sushi and 4 sushi domains, while different
specific reverse primers were used for each of these. Gel electrophoresis
provided confirmation of the successful amplification of genes encoding
1st sushi domain of protein B5R (A) and all 4 sushi domains of B5R protein
(B). Simulation run on Snapgene corresponds to the results obtained in the
lab: 290 bp of PCR product encoding 1st sushi and 756 bp of 4th sushi of
B5R protein.

![](https://static.igem.wiki/teams/4389/wiki/results/gel1.png)![](https://static.igem.wiki/teams/4389/wiki/results/sim1.jpg)

**Figure 1** Results for gel electrophoresis of PCR products
A: Amplified PCR product encoding 1st sushi domain of B5R
protein
B: Amplified PCR product encoding all 4 sushi domains of B5R
protein.
 On the right, the simulation of gel electrophoresis by
SnapGene.

Restriction digestion of pET23a plasmid was performed using restriction
enzymes NdeI and XhoI. Figure 2 gel electrophoresis shows the successfully digested pET23a plasmid,
as lighter fragments have uniformly separated from the double-digested
linearized pET23a plasmid.

Both sequences encoding 1st sushi domain and 4 sushi domains were
double-digested using restriction enzymes NdeI and XhoI, after which PCR
purification of both restriction digestion products was performed.

![](https://static.igem.wiki/teams/4389/wiki/results/gel2.jpg)![](https://static.igem.wiki/teams/4389/wiki/results/sim2.png)

**Figure 2** Results for gel electrophoresis of pET23a plasmid double-digested
by NdeI and XhoI
A - H: Products of restriction digestion of pET23a plasmid.
 On the right, the simulation of gel electrophoresis by
SnapGene.

*E. coli DH5* cells were transformed with the ligation products of
double-digested pET23a plasmid and sequences encoding 1st sushi domain
(Figure 3A) and 4 sushi domains (Figure 3B). For the positive control, cells were transformed with the pUC control
plasmid (Figure 3C). For the negative control, the cells were not transformed prior to
plating (Figure 3D). The cells were grown in a selective medium of Luria Bertani agar and
Ampicillin (100 mg/mL). Plasmid extraction was performed to amplify the
number of ligation products, which are later to be used to transform *E. coli BL-21(DE3)* cells for the expression of protein B5R.

![](https://static.igem.wiki/teams/4389/wiki/results/plates1.png)

**Figure 3** Transformation of *E. coli DH5α*  cells with ligation products: A) *E. coli DH5α*  cells transformed with ligation product of pET23a plasmid and
sequence encoding 1st sushi domain B) *E. coli DH5α*  cells transformed with ligation product pET23a plasmid and sequence
encoding 4 sushi domains C) Positive control: *E. coli DH5α*  cells transformed with pUC control plasmid only D) Negative
control: Non-transformed *E. coli DH5α*  cells.

Colony PCR was performed to screen bacterial colonies for a successful
construct. Colonies were obtained from plates with *E. coli DH5α* transformed with corresponding ligation products. Since we are using the
T7-based expression vector system, forward and reverse T7 primers were
used in the Colony PCR. Colony PCR products of pET23a ligated with the sequence encoding
either 1st sushi domain (Figure 4D, E) or 4 sushi domains (Figure 4F, G) travel less distance due to the presence of T7 primers, and therefore
appear higher in the gel than control runs (Figure 4A, B). Therefore, both *E. coli DH5α* colonies (pET23a + 1st sushi domain) and only the first *E. coli DH5α* colony (pET23a + 4 sushi domains) have been identified for successful
ligation products. The F colony PCR product was sent to sequencing.

![](https://static.igem.wiki/teams/4389/wiki/results/gel3.jpg)

**Figure 4** Results for gel electrophoresis of colony PCR products
A: PCR product encoding 1st sushi domain of B5R protein
B: PCR product encoding all 4 sushi domains of B5R protein
C: pET23a plasmid used as a control
D: Colony PCR product of pET23a + 1st sushi domain sequence
E: Colony PCR product of pET23a + 1st sushi domain
F: Colony PCR product of pET23a + 4 sushi domains
G: Colony PCR product of pET23a + 1st sushi domains

Colony PCR product of pET23a ligated with the sequence encoding 1st sushi
domain was repeated for better results. According to Figure 5, the colony PCR products in wells B, C, D, and F appear higher than the
control sample (A). The colony PCR products of pET23a ligated with
sequence encoding 1st sushi domain obtained from the successful three (B,
C, D) colonies were sent to DNA sequencing analysis.

![](https://static.igem.wiki/teams/4389/wiki/results/gel4.jpg)

**Figure 5** Results for gel electrophoresis of colony PCR products
A: PCR-amplified and purified nucleic acid sequence encoding 1st sushi
domain of B5R protein
span class="c1">B - F:  Colony PCR product of pET23a + 1st sushi domain (Figure 6A)

*E. coli BL-21(DE3)* cells were transformed with the amplified ligation products of
double-digested pET23a plasmid and sequences encoding 1st sushi domain
(Figure 6A) and 4 sushi domains (Figure 6B). For the positive control, cells were transformed with the pUC control
plasmid (Figure 6C). For the negative control, the cells were not transformed prior to
plating (Figure 6D). The cells were grown in a selective medium of Luria Bertani agar and
Ampicillin (100 mg/mL).

![](https://static.igem.wiki/teams/4389/wiki/results/plates2.png)

**Figure 6** Transformation of *E. coli BL-21(DE3)* cells with ligation products: A) *E. coli BL-21(DE3)* cells transformed with ligation product of pET23a plasmid and a
sequence encoding 1st sushi domain B) *E. coli BL-21* cells transformed with ligation product pET23a plasmid and a
sequence encoding 4 sushi domains C) Positive control: *E. coli BL-21(DE3)* cells transformed with pUC control plasmid only D) Negative
control: Non-transformed *E. coli BL-21(DE3)* cells

#### Sequencing

3 samples (reverse and forward for each colony) with 1st sushi domain
and 1 sample with 4 sushi domains were sent for DNA sequencing analysis,
based on the colony PCR results.

**Figure 7** shows the sequencing chromatogram with results from the sequencing
run of 1st sushi domain DNA. From the chromatogram, it can be seen that
the results are clear, with evenly-spaced peaks and no significant
baseline noise.

![](https://static.igem.wiki/teams/4389/wiki/results/seq1.png)

**Figure 7**. Part of the sequencing Chromatogram of reversed 1st sushi domain from
colony A.

The sequencing results were checked for alignment with the original
plasmid DNA for the presence of 1st sushi and all 4 sushi domains’
parts in the colony's DNA for further plasmid extraction. As can be
seen from Figure 8 all samples have aligned with the original DNA sequences encoding
1st sushi and all 4 sushi domains. The sequencing chromatogram confirms
the proper cloning of 1st sushi and all 4 sushi domains into the pET23a
plasmid.

![](https://static.igem.wiki/teams/4389/wiki/results/seq2.png)

****Figure 8****. Sequence alignment with plasmid DNA for 1st sushi and all 4 sushi
domains. Image created in SnapGene.

## Protein expression

#### SDS-PAGE electrophoresis

For the test expression was checked 48 different conditions for the
negative control, 1st sushi domain, and all 4 sushi domains in order to
determine the optimal: incubation time (2h, 4h, 6h, and overnight (ON));
temperature (19°C, 37°C, 37°C); IPTG concentration (0mM, 0.2mM, 0.5mM, 1mM) (Figure 9). After expression, bacteria were centrifuged and pellets were resuspended
in a lysis buffer with freeze-thaw cycles, followed by homogenization. The
presence of B5 was tested using Coomassie blue 15% SDS-PAGE.

The mass of the 1st sushi domain of  B5 protein is ~6 kDa. Despite
the positive results from sequencing and a wide range of test conditions,
the expression of the protein was not observed (Figure 9). Smaller IPTG concentrations (0 mM and 0.2 mM) were not checked due to
lack of time, but it is predicted that there is less probability of
protein expression without IPTG induction.

![](https://static.igem.wiki/teams/4389/wiki/results/sds1.png)

**Figure 9**. SDS-PAGE analysis of B5 1st sushi domain protein. A)19°C. Lane 1-
2h and 0.5 mM IPTG. Lane 2- 4h and 0.5 mM IPTG. Lane 3-6h and 0.5 mM IPTG.
Lane 4- ON and 0.5 mM IPTG. Lane 5- absent, Lane 6- 2h and 1 mM IPTG. Lane
7- 4h and 1 mM IPTG. Lane 8-6h and 1 mM IPTG. Lane 9- ON and 1 mM IPTG. B)
same lanes but at 30°C. C) same lanes but at 37°C, Lane 5-
control

The mass of all 4 sushi domains of B5 protein is ~25 kDa. The expression
of these domains also was not observed (Figure 10). The possible reasons for the problem with expression might include the
following: the expression system is not working (promoter could be
defective); occurrence or absence of the stop codon; problems with protein
folding in the bacterial cytoplasm.

![](https://static.igem.wiki/teams/4389/wiki/results/sds2.png)

**Figure 10**. SDS-PAGE analysis of B5 all 4 sushi domains protein. A)19°C. Lane
1- 2h and 0.5 mM IPTG. Lane 2- 4h and 0.5 mM IPTG. Lane 3-6h and 0.5 mM
IPTG. Lane 4- ON and 0.5 mM IPTG. Lane 5- absent, Lane 6- 2h and 1 mM
IPTG. Lane 7- 4h and 1 mM IPTG. Lane 8-6h and 1 mM IPTG. Lane 9- ON and 1
mM IPTG. B) same Lanes but at 30°C. Lane 5- control C) same lanes but
at 37°C.

In contrast to the B5 protein, the other 3 proteins were successfully
expressed using the same methods. An example of the successful expression
of the L1 protein can be observed in Figure 11, the mass of the L1 is ~27 kDa and the highly concentrated accumulation
of the protein near this mass can be seen in lanes 2 and 8 in red boxes.

![](https://static.igem.wiki/teams/4389/wiki/results/sds3.jpg)

**Figure 11**SDS-PAGE analysis of L1 protein. IPTG concentration- 1 mM, temperature
30°C, 2h of incubation

#### Western blot

The Western blot was conducted for several IPTG-induced samples of B5 1st
sushi and all 4 sushi domains. Despite the small-expression shown on the
SDS-PAGE, the western blot showed that the sample cells have not expressed
the required parts of the B5 protein - 1st sushi and all 4 sushi domains.
The expression was successful for the other 3 recombinant proteins A27,
A33, and L1. Figure 12, 13, and 14 represent the western blot of samples taken from SDS-PAGE analysis
for L1, A27, and A33 proteins, respectively. Results from Western blots
indicate that proteins L1, A27 and A33 were properly folded and
successfully bound to their antibodies, thus antibodies can be used for
biosensor functionalization.

![](https://static.igem.wiki/teams/4389/wiki/results/wb.png)

**Figure 12**. Western blot analysis of L1 protein

![](https://static.igem.wiki/teams/4389/wiki/results/wb2.png)

**Figure 13** Western blot analysis of A27 protein

![](https://static.igem.wiki/teams/4389/wiki/results/wb3.png)

**Figure 14** Western blot analysis of A33 protein

## Aptamer design

We created aptamer sequences for L1, A27, and A33 proteins using an
open-source software MAWS:

Table 1. DNA aptamer sequences for L1, A27, and A33

|  |  |  |
| --- | --- | --- |
| Protein | DNA aptamer sequence | Aptamer ID |
| L1 | ATCGTGAGGAAGCGGCGGGA | L1\_1 |
|  | ATCGTGAGGAAGCG | L1\_2 |
|  | GCGGGGGGGTGCGAAGGGG | L1\_3 |
|  | GCGGGGGGGTGCGAA | L1\_4 |
|  | ATCGTGAGGAAGCGGCGGGATTTGG | L1\_5 |
| A27 | GGAAGGGGGGTGGCCGGCGA | A27\_1 |
|  | GTAGTCGGGGTAGCCGGGAG | A27\_2 |
|  | GGTAGTAAGGCGGGGATAGA | A27\_3 |
|  | GGTAGTAAGGCGGG | A27\_4 |
|  | GGAAGGGGGGTGGCC | A27\_5 |
| A33 | GAGGGGCTGGGGTTGGTTTG | A33\_1 |
|  | GAGGAGGGACTGGGGGGGAG | A33\_2 |
|  | GUGGGCAGGGUAAA | A33\_3 |
|  | GTGGGCAGGGTAAAG | A33\_4 |
|  | GAGGAGGGACTGGGG | A33\_5 |

To make optic fiber biosensors with aptamers, we attached a sequence
‘TTTTT’ to the 5’ of the DNA sequence, so aptamers can
bind to the organic layer on the optic fibers.

And as we had limited time, we made 3D structures for aptamers of only L1
(Figure 15 and 16), using a method described in [Oliveira et al.](https://www.google.com/url?q=https://journals.plos.org/plosone/article?id%3D10.1371/journal.pone.0264701&sa=D&source=editors&ust=1665577532860004&usg=AOvVaw26mRrYBzkqT2uPQxSnErQx) and the software [dnaTurner](https://www.google.com/url?q=http://link_to_gitlab&sa=D&source=editors&ust=1665577532860266&usg=AOvVaw2hCfvCi_Pi0XciLD6gVE-k) that we hacked (see [software page](https://www.google.com/url?q=https://2022.igem.wiki/nu-kazakhstan/software&sa=D&source=editors&ust=1665577532860427&usg=AOvVaw2y4tcRJWt6VB24f9VY-YMh) for more information).

![](https://static.igem.wiki/teams/4389/wiki/results/image5.png)

**Figure 15**3D structure of a DNA aptamer of L1 (ID: L1\_1)

Then to check how well they bind with L1, we docked the 3D structures of
aptamers and the protein in the [HDOCK](https://www.google.com/url?q=http://hdock.phys.hust.edu.cn/&sa=D&source=editors&ust=1665577532860863&usg=AOvVaw2ZGYM2AxEEUX8X_x6Whqqh)web server, and used the [PLIP](https://www.google.com/url?q=https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index&sa=D&source=editors&ust=1665577532861037&usg=AOvVaw0IMRg7g7TeXgPi1wzh-LI5)web server to analyze the docking model with the least binding energy, and ordered the
two aptamers forming the most number of hydrogen bonds for further in-vivo
tests (aptamers L1\_1 and L1\_5).

![](https://static.igem.wiki/teams/4389/wiki/results/image24.png)

|  |  |  |
| --- | --- | --- |
| Docking score | Confidence score | Ligand rmsd (Å) |
| -296.11 | 0.9489 | 41.99 |

**Figure 16** A docking simulation model for L1 and his aptamer (ID: L1\_1)

Table 2. Data of aptamer-L1 interactions

|  |  |  |
| --- | --- | --- |
| Aptamer ID | Docking Confidence Score (via HDOCK) | Number of hydrogen bonds in a docking simulation (via PLIP) |
| L1\_1 | 0.9489 | 15 |
| L1\_2 | 0.8577 | 7 |
| L1\_3 | 0.8806 | 12 |
| L1\_4 | 0.8286 | 5 |
| L1\_5 | 0.8493 | 20 |

## Testing optic fiber biosensors on antigen detection

#### Overview

We conducted tests with two types of optic fiber biosensors: biosensors
functionalized with single antibodies and multiple antibodies (multiplex
biosensors). Biosensors with single antibodies have been tested for
detection of their antigen in PBS solution, while the multiplex sensors
were tested both in PBS and sewage water.

#### Biosensors with single antibodies

Biosensors, made via functionalizing optic fiber ball resonators with
single antibodies, successfully responded to the presence and change of
concentrations of target antigens up to attomolar concentrations and
proved specific to their target antigens.

We functionalized 3 optic fiber ball resonators with antibodies of L1,
A27, and A33 proteins and used one optic fiber ball resonator
functionalized without antibodies as a control, and immersed them in a
solution with the proteins L1, A27 and A33 dissolved in PBS. We started at
the 10 attoMolar concentration of proteins and increased by the power of
10, up to 10 nanomolar concentration. The sensor responses were recorded
10 minutes of immersing in a solution of concentration, and the
measurements for one sensor were consecutive.

Compared to the control sensors, outputs of the optic fiber biosensors
functionalized with single antibodies represented as their amplitudes,
change as a function of the concentration of antigens in PBS (Figure 17). The graphs also show the average and standard deviation of three
measurements, and the Limit of Detection (3ylowest + δmax).The sensors functionalized with single antibodies showed a
logarithmically linear trend with R2 > 0.90, and the range of LOD of sensors to detect vaccinia virus proteins were
from 0.1pM to 1fM. The tests have not been tested for repeatability and
require further validation, however they show a good potential to detect
viral antigens in low concentrations with high confidence.

![](https://static.igem.wiki/teams/4389/wiki/results/image17.png)

**Figure 17** Response of the optic fibers functionalized with antibodies of (a) L1,
(b) A33 and (c) A27, and (d) the control optic fiber as a function of
concentrations of L1, A27, and A33 proteins in PBS

Figure 18 illustrates how the response of optic fibers is usually obtained -
as amplitudes of the sensor at a range of wavelengths and shows how it
changes at different concentrations.

![](https://static.igem.wiki/teams/4389/wiki/results/image22.png)

**Figure 18**Spectra showing the change in the amplitudes of optic fibers
functionalized with antibodies of L1 in different concentrations of the
antigens L1, A27, and A33

To test for the specificity, sensors functionalized with L1, A33 and A27
were immersed in solutions with high concentrations of the target antigen,
and Chemokine ligand 5 (CCl5), Interleukin 4 (IL4) and Thrombin proteins
were used as Controls. The sensors show increased responses (shown in
terms of Refractive Index Unit,) to their corresponding antigens than to
other compounds, indicating their high specificity to the target antigens
(Figure 19).

![](https://static.igem.wiki/teams/4389/wiki/results/image21.png)

**Figure 19**Specificity of the optic fibers functionalized in detecting the target
protein. a- biosensor functionalized with anti-L1 antibodies, b- biosensor
functionalized with anti-A27 antibodies, c- biosensor functionalized with
anti-A33 antibodies.

#### Testing Multiplex biosensors in PBS and sewage water

Multiplex biosensors were observed to respond to the presence of antigens
and their change in concentration both in PBS and in sewage water
samples.

The optic fiber ball resonators were functionalized with three proteins
simultaneously, making a biosensor that can detect several antigens, and
then were tested with the same method in PBS and sewage water. The tests
in PBS were conducted to observe if the antibodies on the surface of the
sensors bind to the antigens in solutions where external interference is
minimized. Then the sensors were tested in synthetic sewage water samples
to detect target proteins in a more challenging and interference-rich
environment, to test the capabilities of the sensor to work in medium-rich
environments.

![](https://static.igem.wiki/teams/4389/wiki/results/image12.png)

**Figure 20**Response of (a) the multiplex sensor and (b) the control optic fiber, in
solutions of different concentrations of L1, A27, and A33 proteins in
PBS

![](https://static.igem.wiki/teams/4389/wiki/results/image20.png)

**Figure 21**Amplitude change of multiplex sensors (a and b) and control sensor (c) as
a function of the concentration of the antigens in sewage water

The multiplex sensors showed a log-linear trend with R2 > 0.92 in PBS and R2 > 0.88 in sewage water samples (Figures 20 and 21). The Limit of Detection for multiplex sensors were in the range of 1fM
and 1pM in sewage water samples, indicating that the multiplex sensors can
detect target antigens in low concentrations with high confidence (Figure 21).

![](https://static.igem.wiki/teams/4389/wiki/results/image7.png)

**Figure 22**Spectra showing the change in the amplitude of the multiplex sensor to
different concentrations of the antigens L1, A17, and A33

![](https://static.igem.wiki/teams/4389/wiki/results/image8.png)

**Figure 23**Spectra showing the change in amplitude of a multiplex optic fiber
biosensor to different concentrations of antigens in sewage water

Overall, the optical fiber ball resonators functionalized with single
antibodies were able to detect vaccinia virus proteins in low
concentrations with high specificity in PBS. The multiplex biosensors have
shown the ability to detect vaccinia virus proteins in low concentrations
both in PBS and sewage water samples.

For a more thorough interpretation of the results, please see the [Proof of Concept](https://www.google.com/url?q=https://2022.igem.wiki/nu-kazakhstan/proof-of-concept&sa=D&source=editors&ust=1665577532872754&usg=AOvVaw2i-5ZMhqWYZRba-m6XqVR8) page.

## Successes and failures

#### Successes

* Designed B5 1st and all 4 sushi constructs
* Expressed L1, A27, A33 properly folded and bound to their
  antibodies
* Optic fiber biosensors detect the presence of target proteins in PBS
  solution and sense changes in concentrations of the target
  proteins.
* Optic fiber biosensors detect the presence of target proteins in sewage
  water and sense changes in concentrations of the target proteins.
* Optic fiber biosensors showed high sensitivity to target
  antigens.
* We designed aptamers for L1, A27, and A33 using MAWS software, and
  modeled and analyzed their interactions in-silico.

#### Failures

* The B5’s 1st and all 4 sushi domains were not expressed due to
  either defect expression system, occurrence or absence of stop codon, or
  improper folding in the cytoplasm.
* Designed aptamers have not been tested in the laboratory for complete
  verification of their binding with the target proteins.
* In the process of implementing our project, the machine that makes ball
  resonators from ordinary optic fibers broke, and thus, because we
  couldn’t make new optic fiber biosensors, we have been limited in
  the number of sensors and experiments on the sensors we can conduct,
  which to a noticeable extent constrained the quality of conclusions we
  can make. For example, it is hard to elaborate on the repeatability of
  the sensors because we cannot make new sensors to test.

## Future tests

* We started the cloning with another plasmid for B5 expression - pet22b
  with the inserted signal sequence for Sec (secretion system) which
  enables secretion within the periplasm. However, due to the lack of time
  and long primer delivery to Kazakhstan, we were not able to start
  testing for the expression. So, we are planning to test all possible
  solutions for B5 protein expression.
* We plan to test the multiplex biosensor in real environmental
  conditions to have reliable results about the duration and stability of
  the biosensor.
* In the future we plan to order and test aptamers for L1, A27, A33, and
  B5 (if successfully expressed).

## Conclusion

Our multiplex biosensor based on optic-fiber functionalized with
antibodies for 3 vaccinia virus proteins (A27, A33, L1) demonstrated the
ability to detect target proteins with high specificity,
concentration-dependent change and stability of response over time both in PBS and sewage water. We
successfully designed aptamers for the desired proteins, but due to a lack
of time, we did not have time to test them on a fiber-optic sensor for
protein detection. Also, due to a defective or inappropriate plasmid
system that we have chosen or due to the toxicity of the protein to BL-21
E.coli, the B5 protein was not synthesized, so the biosensor was not
functionalized for this protein. Thus, we need to test whether the
insertion of the Sec system into the B5 sequence and the usage of
different plasmids will allow the expression of B5.