iGEM 2025 — Team ccu-taiwan

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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# Notebook

## Notebook

✕

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{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
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# Engineering

* [Introduction](#Introduction)
* [Finding the Best Condition of Protein Expression and Purification in *E. Coli*](#Finding)

+ [Summarize](#Summarize1)

* [Changing the Expression System to HEK293T for Best Condition of Protein Expression and Purification](#Changing)

+ [Summarize](#Summarize2)

* [Reference](#Reference)

## Introduction

In order to immobilize sRAGE onto collagen-containing patches, we aimed
to create a fusion protein that contains both sRAGE and CBD. In addition,
we decide to keep the flexibility of this fusion protein in other
application. Therefore, we designed the gene blocks containing
sRAGE-EGFP-6xHis-Npu-DnaEN (sRAGE-IntN for short),
Npu-DnaEC-CWE-6xHis-CBD (IntC-6xHis-CBD for short) and
6xHis-Npu-DnaEC-CWE-CBD (6xHis-IntC-CBD for short), because intein
splicing not only enables the formation of the fusion protein but also
provides greater flexibility for the use of CBD. At the early stage of
the project, we planned to express the target proteins in large
quantities using *E. coli*. However, after team experiments, we found that
the expressed proteins are not soluble and folding properly. We then
shifted to mammalian HEK293T cells. It was known that sRAGE is
N-glycosylated in mammalian cells, and deletion of N-glycan increases the
sRAGE affinity to AGE. Therefore, we ultimately decided to express the
mutated sRAGE (N25Q and N81Q) -IntN and CBD-IntC in mammalian
293T cells.

## Finding the Best Condition of Protein Expression and Purification in *E. Coli*

To express our target protein, we designed four gene blocks and used the
pET15b to construct our expressing plasmids. Each construct was then
transformed into BL21 *E. coli* for protein expression.

**Building constructs for *E. coli***

* **Design & Build** – Design and build constructs:

  The four gene blocks include sRAGE-IntN, IntC-6xHis-CBD,
  6xHis-IntC-CBD, and EGFP-CBD (Figure 1). The gene block
  IntC-6xHis-CBD differs from 6xHis-IntC-CBD in the way of order of
  intein and 6xHis tag, in case the 6xHis affects the intein splicing.
  The gene block EGFP-CBD serve as reporter for Dry Lab to detect the
  CBD anchorage on collagen layer.
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-1-a-new.webp)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-1-b-new.webp)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1203/figure-1-c.avif)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1203/figure-1-d.avif)

  ▲ Figure 1: (A) IntC-6xHis-CBD (B) 6xHis-IntC-CBD (C) EGFP-CBD (D) sRAGE-IntN
* **Test:**

  After successfully assembling the constructs and amplifying the
  plasmids in DH5α, we transformed plasmids sRAGE-IntN, IntC-6xHis-CBD,
  6xHis-IntC-CBD, and EGFP-CBD into BL21 *E. coli* for protein
  expression. Initially, we induced the target proteins at 37°C with
  1 mM IPTG for 2, 4, and 6 hours, but the target proteins were all
  found in the insoluble pellet. As shown in following figure
  6xHis-IntC-CBD proteins were all found in the insoluble pellet
  (Figure 2).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-2.avif)

  ▲ Figure 2: 6xHis-IntC-CBD induced at 37°C with 1 mM IPTG for 0, 2, 4, 6 hours
* **Learn** – The PBS based lysis buffer and sonication may not
  efficiently extract target protein:

  The decreases of induction temperature and IPTG concentration may
  increase the solubility of target protein by slowing down the protein
  production rate. Therefore, we decided to try different lysis
  buffers, and adjust the induction temperature and IPTG
  concentration.

**Replaced lysis buffer & induced condition**

* **Design & Build** – Prepare three different lysis buffers, lower the induction temperature, and reduce the concentration of IPTG:

  To ensure complete cell lysis, we used three different lysis buffers:
  PBS (1 mM PMSF, 0.1% Triton), PBS (1 mM PMSF, 0.5% Triton), and B-PER
  (1 mM EDTA) to lyse the cells. Additionally, we lowered the induction
  temperature to 20°C and reduced the IPTG concentration to ensure
  proper protein folding and normal expression.
* **Test:**

  The three different lysis buffers did not increase the solubility of
  induced protein. As shown in the Figure, sRAGE-IntN was still found
  in the pellet (Figure 3-4).Decreasing the induction temperature to
  20°C also did not enhance the solubility of target protein in the
  supernatant (Figure 5). Finally, we tested the protein induction by
  0.5 mM, 0.25 mM, and 0.125 mM IPTG. As shown in figure,
  IntC-6xHis-CBD remained in the pellet (Figure 6).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-3.webp)

  ▲ Figure 3: sRAGE-IntN induced at 37°C with 1 mM IPTG for 2 hours and lysed in PBS (1 mM PMSF, 0.1% Triton).

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-4.avif)

  ▲ Figure 4: sRAGE-IntN induced at 37°C with 1 mM IPTG for 6 hours and lysed in PBS (1 mM PMSF, 0.5% Triton), and B-PER (1 mM EDTA).

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-5.avif)

  ▲ Figure 5: sRAGE-IntN induced at 20°C with 1 mM IPTG for 0, 2, 4, 6 hours

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-6.avif)

  ▲ Figure 6: IntC-6xHis-CBD induced with 0, 1, 0.5, 0.25 mM IPTG for 6 hours.
* **Learn** – The protein is trapped in the inclusion body:

  Based on attempts with different lysis buffers, we can rule out the
  possibility of incomplete cell lysis. Regardless of whether we
  lowered the induction temperature or reduced the IPTG concentration,
  the target protein was still found in the inclusion bodies.
  Therefore, we decided to refer to the relevant literature on inteins
  to explore other methods that might allow the target protein to be
  released from the inclusion bodies into the supernatant during
  lysis.

**Urea denatured protein**

* **Design & Build** – Denature proteins using 8M urea:

  To solubilize the target protein, we referred to the experimental
  procedures from the literature (Zettler, Schütz, & Mootz, 2009) and
  used the same lysis buffer as in the paper, buffer A-urea (50 mM
  Tris/HCl at pH 8.0, 300 mM NaCl, 8 M urea, 20 mM imidazole).
* **Test:**

  Showing sRAGE-IntN as an example, 8M urea buffer could solubilize target proteins (Figure 7).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-7.webp)

  ▲ Figure 7: sRAGE-IntN induced at 20°C with 1 mM IPTG for 6 hours and lysed in buffer A-urea (50 mM Tris/HCl at pH 8.0, 300 mM NaCl, 8 M urea, 20 mM imidazole).
* **Learn** – The effect of 8M urea is significant; the proteins are all in the supernatant, allowing for protein purification:

  Buffer A-urea dissolves the proteins in the inclusion bodies into the
  supernatant, including the target protein, allowing the experiment to
  proceed toward protein purification.

**Protein purification**

* **Design & Build** – Using Ni-IDA resin for small-scale protein purification:

  Since all the gene blocks are designed with a His tag, the target
  protein can be easily purified using immobilized nickel ions.
  Therefore, we first performed a small-scale protein purification
  using Ni-IDA resin to confirm that the target protein could be
  purified.
* **Test:**

  The SDS-PAGE analysis result indicated that the target protein was
  successfully captured by Ni2+ resin. However, only EGFP-CBD protein
  could be eluted from resin (Figure 8). The sRAGE-IntN,
  IntC-6xHis-CBD, and 6xHis-IntC-CBD protein shows no or slightly
  elution (Figure 9).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-8.avif)

  ▲ Figure 8: EGFP-CBD is purified by NI-IDA resin.

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-9.avif)

  ▲ Figure 9: sRAGE-IntN is purified by NI-IDA resin.
* **Learn** – Elution buffer doesn’t have enough strength to elute the target proteins:

  Except for EGFP-CBD, the elution results of the other proteins were
  very poor. We believe that the current elution buffer (600 mM
  imidazole) does not have enough strength to elute the target
  proteins.

**Trying different elution buffers for protein purification**

* **Design & Build** – Increase the concentration of imidazole in the elution buffer and lower the pH:

  Since the elution of sRAGE-IntN, IntC-6xHis-CBD, and 6xHis-IntC-CBD
  protein from resin is poor, we increased the imidazole concentration
  in the elution buffer to 1M and 2M, and in addition, we also try an
  acid elution buffer at pH 2.5.
* **Test:**

  sRAGE-IntN, IntC-6xHis-CBD, 6xHis-IntC-CBD protein were eluted using
  three different elution buffers. Both 1M and 2M imidazole slightly
  increase the elution of Ni-IDA resin bound protein (Figure 10). The
  acid elution buffer did not work (Figure 11).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-10.avif)

  ▲ Figure 10: sRAGE-IntN was eluted using 1M and 2M imidazole.

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-11.avif)

  ▲ Figure 11: sRAGE-IntN was eluted at pH 2.5.
* **Learn** – The concentration of the eluted protein is not sufficient:

  Increasing the concentration of imidazole in elution is feasible, but
  the eluted protein concentration is not very high. Therefore, we
  reduced the volume of the elution buffer.

**Increase the concentration of eluted protein**

* **Design & Build** – Reduce the volume of the elution buffer to 200 µl:

  To increase the concentration of the eluted protein, we used an
  elution buffer with 1M imidazole and reduced the volume to 200 µl.
* **Test:**

  According to SDS-PAGE, the protein concentration in the elution
  increased significantly, but most target protein was still trapped on
  the Ni-IDA resin (Figure 12).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-12.avif)

  ▲ Figure 12: sRAGE-IntN was eluted with 1M imidazole in the volumn of 200 µl.
* **Learn** – The presence of inteins may affect protein purification:

  Although the protein concentration in the elution has increased, not
  all of the target proteins were eluted. In addition to continuing to
  search for the optimal protein purification conditions, we also
  suspect that the presence of intein may affect protein purification.
  Because in addition to EGFP-CBD, we also prepared a positive control
  (EGFP-4A-C7 from 2023 CCU-iGEM) (Figure 13.) to confirm that the
  target protein without the intein can be successfully purified.
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-13.avif)

  ▲ Figure 13: EGFP-4A-C7 from 2023 CCU-iGEM was eluted by 600mM imidazole.

**Intein splicing**

* **Design & Build** – Verify whether correct splicing can occur under unpurified conditions and evaluate whether the position of the 6xHis tag affects intein splicing:

  Although the purification of the sRAGE-IntN, IntC-6xHis-CBD, and
  6xHis-IntC-CBD protein was not completely successful, the reference
  suggested that intein splicing can still be performed in 8M urea
  buffer. We conducted two sets of intein splicing reactions under
  the conditions of 37°C for 0, 0.5, and 1 hours. One set is sRAGE-IntN
  and IntC-6xHis-CBD and the other one is sRAGE-IntN and
  6xHis-IntC-CBD.
* **Test:**

  Both set of combination have been successfully spliced at 37°C for 0,
  0.5, and 1 hour (Figure 14-15). However, after splicing, the protein
  bands on the SDS-PAGE are not very clear.
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/newnew/14-a.avif)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/newnew/14-b.avif)

  ▲ Figure 14: (A) 12% SDS-PAGE analysis of sRAGE-6xHis-CBD which was spliced by IntC-6xHis-CBD and sRAGE-IntN (B) 6% SDS-PAGE analysis of sRAGE-6xHis-CBD which was spliced by IntC-6xHis-CBD and sRAGE-IntN

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/newnew/15-a.avif)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/newnew/15-b.avif)

  ▲ Figure 15: (A) 12% SDS-PAGE analysis of sRAGE-6xHis-CBD which was spliced by 6xHis-IntC-CBD and sRAGE-IntN (B) 6% SDS-PAGE analysis of sRAGE-6xHis-CBD which was spliced by 6xHis-IntC-CBD and sRAGE-IntN
* **Learn** – Obtain more discriminative results using Western blot:

  Since the protein bands after splicing are too faint to distinguish clearly,
  we believe that using Western blot can help confirm the splicing results.

**Western blot analysis**

* **Design & Build** – Obtained the results of intein splicing using Western blot:

  To more accurately determine whether the intein splicing of
  sRAGE-6xHis-CBD was successful, we performed a Western blot.
* **Test:**

  According to the Western blot results, splicing between
  IntC-6xHis-CBD and sRAGE-IntN was successful at 37°C for 0 hr, 0.5
  hr, and 1 hr. However, the amount of successfully spliced protein
  between 6xHis-IntC-CBD and sRAGE-IntN appeared to be low, and no
  successful splicing was observed at 0 hr (Figure 16).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/newnew/16.avif)

  ▲ Figure 16: Western blot analysis of two sets of intein splicing
* **Learn** – The position of 6xHis affected intein splicing:

  Under the same conditions, the splicing efficiency of IntC-6xHis-CBD
  and sRAGE-IntN is better than that of 6xHis-IntC-CBD and sRAGE-IntN.
  We speculate that the position of the 6xHis tag affects intein
  splicing.

## Summarize

*E. coli* can successfully express sRAGE-IntN, IntC-6xHis-CBD,
6xHis-IntC-CBD, and EGFP-CBD, but soluble protein can only be obtained
through urea denaturation. Although proteins denatured with urea can
still be purified and undergo intein splicing, we are still concerned
that, aside from the His tag used for purification and intein, most of
the other proteins may be misfolded due to urea denaturation, resulting
in loss of protein functionality. We can verify our concerns based on
the results of EGFP-CBD [[See the result of EGFP-CBD](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D#Experimental3)]. Therefore, to
ensure that we obtain functional proteins, we have decided to switch
the expression system to HEK293T cells.

## Changing the Expression System to HEK293T for Best Condition of Protein Expression and Purification

To overcome the protein expression issues in *E. coli*, we shift to
mammalian cells to expression our proteins.

We then designed five new constructs based on pcDNA3.1 for protein
expression in HEK293T cells. sRAGE-IntN, IntC-6xHis-CBD, 6xHis-IntC-CBD,
and EGFP-CBD are adapted from our original *E. coli*-expressed constructs,
while the fifth was an sRAGE N25Q and N81Q mutant (mut-sRAGE-IntN),
which harbors a higher AGE-binding affinity than sRAGE-IntN.

**Building constructs for HEK293T cells**

* **Design & Build** – Designed and constructed five new pcDNA3.1-based constructs for HEK293T expression:

  Gene blocks of sRAGE-IntN, IntC-6xHis-CBD, 6xHis-IntC-CBD, and
  EGFP-CBD were adapted from our original *E. coli*-expressed version
  (Figure 17). In addition, a mutant construct (mut-sRAGE–IntN)
  carrying N25Q and N81Q substitutions was generated to compare the
  AGE-binding affinity with wild-type sRAGE–IntN (Figure 17).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-17-a-new.webp)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-17-b-new.webp)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-17-c-new.webp)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-17-d-new.webp)

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-17-e-new.webp)

  ▲ Figure 17: (A) sRAGE-IntN(B) mut-sRAGE–IntN (C) IntC-6xHis-CBD (D) 6xHis-IntC-CBD (E) EGFP-CBD
* **Test:**

  To assemble these constructs, we first cloned sRAGE -IntN and mut-sRAGE
  -IntN into pcDNA3.1 backbone using Gibson Assembly but failed.
  We then use BamHI and EcoRI restriction enzyme to clone insert
  into pcDNA3.1. The sequencing result showed an additional sequence
  located upstream of sRAGE (Figure 18).

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-18.avif)

  ▲ Figure 18: The electrogram shows the unexpected 6xHis-T7 tag sequence detected ahead of the sRAGE insert.
* **Learn** – Investigating the impact of unexpected 6xHis–T7 insertion on protein expression:

  Based on the sequencing result, we knew that the unexpected 6xHis–T7
  sequence in the vector eliminated the homologous overlap required for
  Gibson Assembly, leading to assembly failure. Importantly, the
  unintended upstream insertion did not affect the reading frame but
  likely disrupted the signal peptide.

**Protein analysis in HEK293T cells expressed from an incorrect plasmid construct**

* **Design & Build** – Examining the effects of unexpected 6×His–T7 Tag in sRAGE-IntN secretion:

  We transfected the plasmid into HEK293T cells and examined expression
  using fluorescence microscopy and immunocytochemistry (ICC). To
  further assess whether the protein was secreted, we use Western blot
  to performed on both cell lysates and culture supernatants.
* **Test:**

  Fluorescence microscopy showed normal expression of the target
  protein in cells with green fluorescence (Figure 19), and ICC further
  showed the cytoplasmic location in HEK293T cells (Figure 20). For the
  localization, Western blot confirmed that the protein was at the
  expected size and there wasn’t any target protein secret out of cells
  (Figure 21).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-19.webp)

  ▲ Figure 19: The fluorescence image of sRAGE-IntN protein expression in HEK293T cells.

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-20.avif)

  ▲ Figure 20: Immunocytochemistry analysis of 6xHis-T7-sRAGE-IntN protein in 293T cells.

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-21.avif)

  ▲ Figure 21: The Western blot analysis of 6xHis-T7-sRAGE-IntN protein expression in HEK293T. (A) Without any protein in the cell cultured medium. (B) With target protein inside the soluble supernatant of cell lysate. S: soluble supernatant P: insoluble pellet.
* **Learn** – The extra peptide at N-termal of signal peptide blocks
  secretion without affecting expression or folding.

  The signal peptide
  of sRAGE appears highly sensitive to upstream extensions, and any
  addition before it can block secretion. Based on this finding, we
  proceeded to redesign the construct by removing the unintended
  T7–6xHis segment.

**Remove unexpected sequence and check**

* **Design & Build** – Vector reconstruction with HindIII/EcoRI:

  To restore proper secretion, we reconstructed the vector by removing
  the unwanted upstream sequence. A new primer containing a HindIII
  site complementary to the insert was designed to replace the sequence
  via overlap extension PCR. Then, we used HindIII and EcoRI digestion
  to clone sRAGE -IntN, mut-sRAGE -IntN, IntC-6xHis-CBD,
  6xHis-IntC-CBD, and EGFP-CBD into pcDNA3.1, and sent the constructs
  for sequencing.
* **Test:**

  Sequencing confirmed that the unwanted upstream sequence had been
  successfully removed and that the gene blocks were correctly inserted into
  the pcDNA3.1 vector. The cloning results for sRAGE–IntN are presented in
  Figure 22.

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-22.avif)

  ▲ Figure 22: The electrogram shows the boundary between pcDNA3.1 and sRAGE-IntN.
* **Learn** – Correction of construct errors by PCR enabled successful protein expression:

  This result demonstrates that incorrect sequences in a construct can
  be corrected through PCR using designed primers. Therefore, with the
  corrected constructs, we can proceed to transfect cells and express
  the intended protein.

**Protein expression and purification of sRAGE -IntN and mut-sRAGE -IntN**

* **Design & Build** – The proteins secreted in cuture medium were purified with Ni-IDA resin and verified by Western blot:

  We transfected the sRAGE -IntN and mut-sRAGE -IntN plasmids into
  HEK293T cells and confirmed protein expression by fluorescence
  microscopy. The cell culture medium containing secreted proteins
  was stored at –80 °C to avoid degradation. For each purification,
  we used Ni-IDA resin same protocol as for *E. coli* to express proteins
  and use Western blotting to assess purification success.
* **Test:**

  Fluorescence microscopy confirmed normal expression of the target
  protein in Figure 23 and Western blot results verified that the
  protein was secreted into the culture medium at the expected size.
  However, the protein failed to bind to Ni2+ resin and was detected
  entirely in the flow-through (Figure 24).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-23.webp)

  ▲ Figure 23: The immune-fluorescence image of sRAGE-IntN with 6xHis-T7 tag protein expression in HEK293T cells.

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-24.avif)

  ▲ Figure 24: The Western blot analysis of sRAGE-IntN protein purification by NI-IDA resin.
* **Learn** – Insufficient binding time likely reduced binding efficiency:

  Western blot analysis confirmed that the transfected protein
  exhibited the expected size, but its binding efficiency to the
  resin was relatively low. Therefore, we guessed that either the
  binding time was insufficient or that imidazole in the buffer was
  interfering with His-tag interaction.

**Optimization of binding and elution conditions**

* **Design & Build** – Binding the protein overnight in PBS without imidazole, then washing and eluting with high-imidazole buffers:

  To improve purification efficiency, the storage buffer was replaced
  with PBS lacking imidazole, and the binding step was extended from 1 hour
  to overnight. Additionally, because previous attempts failed to
  efficiently elute the protein, the elution buffer was replaced with PBS
  containing a higher imidazole concentration (1 M).
* **Test:**

  The Western blot analysis revealed that the target protein remained
  bound to the Ni-IDA resin and could not be eluted, even under
  high-imidazole conditions (Figure 25).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-25.avif)

  ▲ Figure 25: The Western blot analysis of sRAGE-IntN purification following overnight binding and elution with 1 M imidazole in PBS.
* **Learn** – Intein-containing proteins from *E. coli* and HEK293T cells were difficult to elute:

  We suspected that the failure to elute was either due to the intein
  domain obstructing His-tag accessibility or to suboptimal buffer
  composition. Since the target protein was not eluted, in addition to
  continuing to search for the optimal protein purification conditions,
  we also suspect that the presence of the intein may affect the
  functionality of the 6xHis tag. Therefore, we used a positive
  control, EGFP-4A-C7 provided by CCU-iGEM 2023, to verify that our
  hypothesis is plausible (Figure 26).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-26.webp)

  ▲ Figure 26: EGFP-4A-C7 from 2023 CCU-iGEM was eluted by 600mM imidazole.

**Expressing IntC-6xHis-CBD, 6xHis-IntC-CBD, and EGFP-CBD protein in HEK293T**

* **Design & Build** – Expression of IntC-6xHis-CBD, 6xHis-IntC-CBD, and EGFP-CBD in HEK293T:

  Following the successful expression of sRAGE-IntN and mut-sRAGE-IntN,
  we proceeded to express IntC-6xHis-CBD, 6xHis-IntC-CBD, and EGFP-CBD
  in HEK293T. Then we used fluorescence microscopy to examine
  expression and performed Western blot to check the size of our
  protein.
* **Test:**

  Fluorescence microscopy confirmed normal expression of the EGFP-CBD
  protein in cells, as indicated by green fluorescence (Figure 27).
  The Western blot analysis further showed that EGFP-CBD was detected
  at the expected size. However, no detectable bands corresponding to
  IntC-6xHis-CBD or 6xHis-IntC-CBD were observed (Figure 28).
  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/figure-27.webp)

  ▲ Figure 27: The immune-fluorescence image of EGFP-CBD protein expression in HEK293T cells.

  ![](https://static.igem.wiki/teams/5854/img/page-img/engineenring/1009/figure-28.avif)

  ▲ Figure 28: The Western blot analysis of IntC-6xHis-CBD, 6xHis-IntC-CBD, and EGFP-CBD.
* **Learn** – Intein-containing constructs may have failed to express

  The absence of signal for IntC–6xHis–CBD and 6xHis–IntC–CBD suggests
  that transfection or expression of these constructs was
  unsuccessful. Further optimization such as adjusting transfection
  conditions will be required to determine whether the lack of
  expression is due to transfection inefficiency, instability of the
  construct, or rapid degradation of the protein.

## Summarize

We redesigned and rebuilt multiple pcDNA3.1 constructs for expression in
HEK293T cells to overcome the limitations observed in *E. coli* expression.
Initial cloning attempts failed due to primer mismatches and the
introduction of an unwanted sequence, but vector reconstruction using
HindIII/EcoRI and overlap extension PCR successfully resolved these
issues, as confirmed by sequencing. The verified constructs were
transfected into HEK293T cells, and protein expression and secretion
were confirmed by fluorescence microscopy and Western blotting. However,
purification using nickel beads revealed inefficient binding and elution.
Optimization with overnight binding and higher imidazole concentrations
improved binding but did not resolve elution, particularly for
intein-containing proteins. These results suggest that either the
intein domain or buffer conditions hindered elution, and further
testing with proteins previously successfully purified will help clarify
the underlying cause.

## Reference

1. Zettler, J., Schütz, V., & Mootz, H. D. (2009). The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. *FEBS letters, 583*(5), 909-914.

{% endblock %}


{% extends "layout.html" %}
{% block title %}Result{% endblock %}
{% block page\_content %}

# Results

* [The sRAGE-CBD Protein Expression in *E. Coli*](#The-sRAGE-CBD)

+ [Overview of gene blocks design for protein expression in *E. coli*](#Overview1)
+ [Experimental result](#Experimental1)

* [The sRAGE-CBD Protein Expression in HEK293T](#HEK293T)

+ [Overview of gene blocks design for protein expression in HEK293T](#Overview2)
+ [Experimental result](#Experimental2)

* [Evaluation of CBD Anchorage on Hydrogel](#Evaluation)

+ [Overview](#Overview3)
+ [Design of the EGFP–CBD construct](#Experimental3)
+ [Expressing EGFP-CBD protein for hydrogel attachment test](#Expressing)

* [Future Work](#Future-Work)
* [Reference](#Reference)

* [Patch Layered Design](#Patch)

+ [Overview](#Overview)
+ [Inspiration](#Inspiration)

* [The Preparation of Collagen-Chitosan Layer](#The-preparation)

+ [The effects of NaOH concentration in chitosan crosslinking](#The-effects)
+ [0.01M NaOH is not suitbale for Collagen and Chitosan Hydrogel crosslink](#0.01M-NaOH)
+ [Adjusting the NaOH concentration and container base area](#Adjusting)
+ [Applying the new condition to Collagen–Chitosan hydrogel crosslink](#Applying)

* [Future Vision](#Future-Vision)
* [Reference](#Reference2)

To address the inflammation caused by the accumulation of advanced glycation
end-products (AGEs), we developed a therapeutic patch named **AGE-Thwart**,
consisting of an sRAGE–CBD fusion protein embedded within a collagen–chitosan
hydrogel matrix.

To systematically validate its functionality, the development of AGE-Thwart was
divided into two major experimental modules: first, the production and analysis
of the functional sRAGE–CBD protein, and second, the preparation and testing
of the collagen–chitosan hydrogel matrix.

Expression and Functional
Analysis of sRAGE–CBD
Preparation and Functional Assessment
of Collagen–Chitosan Hydrogel

## Expression and Functional Analysis of sRAGE–CBD

To systematically evaluate the patch, we divided this sub-project into two parts, each targeting a different experimental goal. The first focused on producing the full sRAGE–CBD fusion protein via intein splicing, ensuring proper expression and integrity. The second used EGFP–CBD fusion protein to independently assess the hydrogel-binding capability of the CBD domain. This two-part approach allowed us to optimize both protein production and functional performance for hydrogel attachment.

## The sRAGE-CBD Protein Expression in *E. Coli*

## Overview of gene blocks design for protein expression in *E. coli*

To anchor sRAGE protein onto the collagen layer of patch and preserve the
flexibility for replacing sRAGE with other effective proteins (e.g. anti-microbial
peptide), we decide to apply the intein system to fuse the collagen binding domain
(CBD) with sRAGE.

Accordingly, the C-terminal of sRAGE protein was fused with Npu-DnaEN, the N-terminal
of split intein, while the N-terminal of CBD was fused with Npu-DnaEC, the C-terminal
of split intein. The peptide sequence at the N-terminal of Npu-DnaEN and C-terminal
of Npu-DnaEC were respectively modified to RGK (sRAGE-RGK-Npu-DnaEN) and CWE
(Npu-DnaEC-CWE-CBD) for efficient splicing (Cheriyan, Pedamallu, Tori, & Perler,
2013). The 6xHis tag was added on sRAGE-RGK-Npu-DnaEN and Npu-DnaEC-CWE-CBD for
protein purification (sRAGE-6xHis-RGK-Npu-DnaEN, 6xHis-Npu-DnaEC-CWE-CBD, and
Npu-DnaEC-CWE-6xHis-CBD).

Finally, we fused the EGFP protein to sRAGE-6xHis-RGK-Npu-DnaEN to monitor the
interaction between sRAGEs and AGEs (sRAGE-EGFP-6xHis-RGK-Npu-DnaEN). The expression
of fusion protein was under the control of Lac promoter.
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/new-result-1.avif)

▲ Figure 1: The gene block design of intein-spliced sRAGE-CBD for *E. coli* expression. (A) The N-terminal fragment or (B) C-terminal fragment of sRAGE-CBD fusion construct. The C-terminal fragment of sRAGE-CBD has two variants, which differ in the order of intein and 6xHis tag.

## Experimental result

**Cloning the designed gene blocks into pET15b**

To express the sRAGE-EGFP-6xHis-RGK-(Npu-DnaEN) (sRAGE-IntN for short),
6xHis-(Npu-DnaEC)-CWE-CBD (6xHis-IntC-CBD for short) and (Npu-DnaEC)-CWE-6xHis-CBD
(IntC-6xHis-CBD for short) protein in *E. coli*, we ordered the codon-optimized gene
blocks from IDT and performed Gibson assembly to insert the gene blocks into pET15b
vector. The sequence was verified by Sanger sequencing before transformation into
BL21 for protein expression (Figures 2, 3 and 4).

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/2.avif)

▲ Figure 2: The electrogram shows the boundary between pET15b and sRAGE-IntN.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/3.avif)

▲ Figure 3: The electrogram shows the boundary between pET15b and IntC-6xHis-CBD.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/4.avif)

▲ Figure 4: The electrogram shows the boundary between pET15b and 6xHis-IntC-CBD.

**Cloning the designed gene blocks into pET15b**

We then induced the expression of sRAGE-IntN by 1 mM IPTG. The induced protein
was harvested at indicated time points (0 hr and 2 hr) at 37℃ (Figure 5).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/5.avif)

▲ Figure 5: The sRAGE-IntN protein induction at 37℃ was shown by Coomassie blue staining of SDS-PAGE analysis.

**The induction of sRAGE-IntN protein in *E. coli* at 20 °C**

Since the induced sRAGE-IntN protein located in the inclusion bodies, we then try to
enhance the solubility by decreasing the induction temperature to 20 °C. The induced
sRAGE-IntN protein was harvested at indicated time points (0 hr, 2hr, 4hr, and 6 hr).
Unfortunately, the expressed sRAGE-IntN protein was still in the insoluble fraction,
suggesting the inclusion body’s location (Figure 6).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/6.avif)

▲ Figure 6: The sRAGE-IntN protein induction at 20℃ was shown by Coomassie blue staining of SDS-PAGE analysis. S: supernatant; P: cell pellet.

**Solubilizing sRAGE-IntN protein from inclusion body by 8M urea**

To solubilize the insoluble sRAGE-IntN protein for subsequent experiments, we
applied the 8 M urea extraction buffer to the insoluble fraction, and confirmed the
efficient extraction (Figure 7).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/7.avif)

▲ Figure 7: The extraction of sRAGE-IntN protein from inclusion body was shown by Coomassie blue staining of SDS-PAGE analysis.

**The preparation of IntC-6xHis-CBD and 6xHis-IntC-CBD proteins.**

Similar to sRAGE-IntN, IntC-6xHis-CBD and 6xHis-IntC-CBD protein were both expressed
in the insoluble fraction, either at 20 °C or 37 °C (Figures 8-11). Therefore, we
applied 8 M urea buffer to solubilize the IntC-6xHis-CBD and 6xHis-IntC-CBD proteins
from pellet fraction (Figures 12 and 13).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/8.avif)

▲ Figure 8: The IntC-6xHis-CBD protein induction at 37℃ was shown by Coomassie blue staining of SDS-PAGE analysis. S: supernatant; P: cell pellet.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/9.avif)

▲ Figure 9: The IntC-6xHis-CBD protein induction at 20℃ was shown by Coomassie blue staining of SDS-PAGE analysis. S: supernatant; P: cell pellet.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/10.avif)

▲ Figure 10: The 6xHis-IntC-CBD protein induction at 37℃ was shown by Coomassie blue staining of SDS-PAGE analysis. S: supernatant; P: cell pellet.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/11.avif)

▲ Figure 11: The 6xHis-IntC-CBD protein induction at 20 ℃ was shown by Coomassie blue staining of SDS-PAGE analysis. S: supernatant; P: cell pellet.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/12.avif)

▲ Figure 12: The extraction of IntC-6xHis-CBD protein from inclusion body was shown by Coomassie blue staining of SDS-PAGE analysis.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/13.avif)

▲ Figure 13: The extraction of 6xHis-IntC-CBD protein from inclusion body was shown by Coomassie blue staining of SDS-PAGE analysis.

**The purification of expressed proteins by His-tag**

To purify sRAGE-IntN, IntC-6xHis-CBD and 6xHis-IntC-CBD protein, we performed Ni-IDA
resin. We first tested a 1-hour binding, followed by washing with PBS containing 100
mM imidazole to remove nonspecific proteins, and then attempted to elute the target
protein with PBS containing 600 mM imidazole (Figures 14-16). The SDS-PAGE analysis
of eluted protein and beads suggested the poor elution efficiency.
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/14.avif)

▲ Figure 14: The purification of sRAGE-IntN protein by Ni-IDA resin was shown by Coomassie blue staining of SDS-PAGE analysis.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/15.avif)

▲ Figure 15: The purification of IntC-6xHis-CBD protein by Ni-IDA resin was shown by Coomassie blue staining of SDS-PAGE analysis.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/16.avif)

▲ Figure 16: The purification of 6xHis-IntC-CBD protein by Ni-IDA resin was shown by Coomassie blue staining of SDS-PAGE analysis.

We next increased the concentration of imidazole in elution buffer to 1 M and 2 M,
and the SDS-PAGE analysis result show a slight improvement of elution (Figure 17).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/17.avif)

▲ Figure 17: The elution of sRAGE-IntN protein by 1M and 2M imidazole was shown by Coomassie blue staining of SDS-PAGE analysis.

We then reduced elution buffer volume to 200 µl. The SDS-PAGE analysis result suggested
the better improvement of elution (lane E1). However, most sRAGE-IntN protein still
bound to the beads (Figure 18).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/18.avif)

▲ Figure 18: The elution of sRAGE-IntN protein by 1M imidazole was shown by Coomassie blue staining of SDS-PAGE analysis.

In addition, we examined whether low-pH elution (0.1M glycine buffer, pH 2.5) works.
However, the recovery was still poor, and substantial amounts of protein were
retained on the resin (Figure 19).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/19.avif)

▲ Figure 19: The elution of sRAGE-IntN protein by low-pH elution buffer was shown by Coomassie blue staining of SDS-PAGE analysis.

Similar to sRAGE-IntN, the elution of IntC-6xHis-CBD and 6xHis-IntC-CBD proteins was
inefficient by 1M imidazole (Figures 20 and 21) or low-pH elution buffer (Figures 22
and 23).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/20.avif)

▲ Figure 20: The elution of IntC-6xHis-CBD protein by 1M imidazole was shown by Coomassie blue staining of SDS-PAGE analysis.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/21.avif)

▲ Figure 21: The elution of 6xHis-IntC-CBD protein by 1M imidazole was shown by Coomassie blue staining of SDS-PAGE analysis.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/22.avif)

▲ Figure 22: The elution of IntC-6xHis-CBD protein by low-pH buffer was shown by Coomassie blue staining of SDS-PAGE analysis.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/23.avif)

▲ Figure 23: The elution of 6xHis-IntC-CBD protein by low-pH buffer was shown by Coomassie blue staining of SDS-PAGE analysis.

**The innate characteristic of intein affects Ni2+ resin mediated purification**

After multiple attempts, we suspected that the strong retention was caused by the
Npu-DnaE intein domain. To support this hypothesis, we performed the His-Tag
purification of protein without intein domain by the same elution buffer, showing
that the elution failure was not due to experimental operation or buffer composition
but specifically related to the presence of the Npu-DnaE intein.

The SDS-PAGE analysis result revealed a distinct band in the elution fraction 1 (E1)
for the EGFP-CBD protein lacking the Npu-DnaE intein, indicating efficient elution
(Figure 24).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/24.avif)

▲ Figure 24: The elution of EGFP-CBD protein by 600mM imidazole was shown by Coomassie blue staining of SDS-PAGE analysis.

Similarly, the SDS-PAGE analysis result showed a distinct band in the elution fraction
1 (E1) of the EGFP-4A-C7 protein without the Npu-DnaE intein (from 2023 CCU-iGEM), indicating
that the protein was efficiently eluted (Figure 25).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/25.avif)

▲ Figure 25: The elution of EGFP-4A-C7 protein (from 2023 CCU-iGEM) by 600mM imidazole was shown by Coomassie blue staining of SDS-PAGE analysis.

**Validating intein activity of sRAGE-IntN, IntC-6xHis-CBD, and 6xHis-IntC-CBD**

The 8M urea buffer unfold most extracted protein. However, the previous report suggested
that the intein splicing would not be affected (Zettler, Schütz, & Mootz, 2009).
Accordingly, we mix cell lysate containing sRAGE-IntN with lysate containing
IntC-6xHis-CBD or 6xHis-IntC-CBD. The mixtures were incubated at 37 °C, and aliquots
were collected at 0, 0.5, and 1 hour, followed by quenching with SDS.

The SDS-PAGE analysis revealed that, after mixing, a new band appeared at the expected
molecular weight position corresponding to either sRAGE-EGFP-6xHis-RGK-CWE-6xHis-CBD
(named sRAGE-2His-CBD for short) or sRAGE-EGFP-6xHis-RGK-CWE-CBD (named sRAGE-His-CBD
for short). The band was more clearly visible on a 6% PAGE gel. However, the band
intensity gradually decreased with longer incubation time, which we speculate was due
to protein degradation resulting from the use of unpurified samples (Figures 26 and
27).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/26a.avif)

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/26b.avif)

▲ Figure 26: The splicing of sRAGE-IntN and IntC-6xHis-CBD at 37 °C was shown by Coomassie blue staining of (A) 12% SDS-PAGE and (B) 6% SDS-PAGE analysis. 1: IntC-6xHis-CBD (12.65kDa); 4: sRAGE-IntN (71.60kDa); 4+1: sRAGE-2His-CBD (68.97kDa)

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/27a.avif)

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/27b.avif)

▲ Figure 27: The splicing of sRAGE-IntN and 6xHis-IntC-CBD at 37 °C was shown by Coomassie blue staining of (A) 12% SDS-PAGE and (B) 6% SDS-PAGE analysis. 1: IntC-6xHis-CBD (12.65kDa); 2: 6xHis-IntC-CBD (12.65kDa); 4: sRAGE-IntN (71.60kDa); 4+2: sRAGE-His-CBD (68.31kDa)

Since Coomassie-stained analysis of the sRAGE-IntN lysate revealed a background
protein with a molecular weight similar to the expected splicing product, we further
performed a specific Western blot assay to verify the presence of the spliced
protein. The results showed that the sRAGE-IntN and IntC-6xHis-CBD mixture exhibited
a distinct band at the expected molecular weight at 0 h, while the sRAGE-IntN and
6xHis- IntC-CBD mixture showed the same result after 0.5 h, indicating that the
sRAGE-IntN and IntC-6xHis-CBD pairing reacted faster and achieved a higher splicing
efficiency than the sRAGE-IntN and 6xHis- IntC-CBD pairing (Figure 28).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/28.avif)

▲ Figure 28: The splicing of sRAGE-IntN and IntC-6xHis-CBD or 6xHis-IntC-CBD at 37 °C was shown by Western blot. 1: IntC-6xHis-CBD (12.65kDa); 2: 6xHis-IntC-CBD (12.65kDa); 4: sRAGE-IntN (71.60kDa); splicing product 4+1: sRAGE-2His-CBD (68.97kDa); splicing product 4+2: sRAGE-His-CBD (68.31kDa)

These results indicated that the intein is functional even in the 8 M urea buffer,
suggesting the concept of intein splicing mediated fusion protein is workable.
However, the intein splicing products were not significant, likely due to the
denaturing conditions of 8M urea. Therefore, we concluded that E. coli expression
may not be optimal for producing soluble, properly folded protein with intein, and
decided to switch to a mammalian expression system to avoid the need for harsh
denaturants and improve protein solubility.

## The sRAGE-CBD protein expression in HEK293T

## Overview of gene blocks design for protein expression in HEK293T

To solve the problem of inclusion body expression of designed protein in *E. coli*,
we decided to shift the expression system to mammalian cell line HEK293T. Since the
RAGE originates from trans-membranous protein, we added signal peptide sequence to
sRAGE-EGFP-6xHis-Npu-DnaEN (sRAGE-IntN for short) for proper expression in HEK293T.
Accordingly, the expressed protein should be harvested from culture medium.
Furthermore, we mutated the N-glycosylation sites (N25Q and N81Q) on sRAGE to deplete
the N-glycan, which increase the binding affinity of sRAGE to AGEs (mut-sRAGE-IntN).
Together with 6xHis-Npu-DnaEC-CWE-CBD (6xHis-IntC-CBD for short), and
Npu-DnaEC-CWE-6xHis-CBD (IntC-6xHis-CBD for short), the gene block of all four
protein were codon-optimized and cloned into pcDNA3.1 by conventional molecular
cloning technique.
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/new-result-9.avif)

▲ Figure 29: The gene block design of sRAGE-IntN, mut-sRAGE-IntN, 6xHis-IntC-CBD, and IntC-6xHis-CBD for HEK293T expression. (A) The N-terminal fragment or (B) C-terminal fragment of sRAGE-CBD fusion construct. The N-terminal fragment of sRAGE-CBD has two variant, including wild type sRAGE and N-glycan deletion sRAGE (mut-sRAGE). The C-terminal fragment of sRAGE-CBD has two variants, which differ in the order of intein and 6xHis tag.

## Experimental result

**The unexpected 6xHis-T7 tags in pcDNA3.1**

The gene blocks of sRAGE-IntN and mut-sRAGE-IntN are first cloned into pcDNA3.1 by
restriction enzyme BamHI and EcoRI. However, the Sanger sequencing verification
indicated the unexpected 6xHis-T7 tag in front of our designed signal peptide
sequence (Figure 30), and further examination showed that this unexpected tag
per-exist in the vector, suggesting the wrong vector map sequence.
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/30.avif)

▲ Figure 30: The electrogram shows the unexpected 6xHis-T7 tag sequence at the 5’ terminal of sRAGE-IntN.

**The cytoplasmic expression of 6xHis-T7-sRAGE-IntN protein**

Since the N-terminal tag would abolish the function of signal peptide and hamper
the secretion of expressed protein, we must re-construct the sRAGE-IntN expressing
vector. However, the 6xHis-T7 tag is inflame with sRAGE-IntN, suggesting that this
construct (6xHis-T7-sRAGE-IntN) could still provide information about the location
and folding of sRAGE-IntN protein. Therefore, we first performed fluorescence
microscope observation. The result showed the positive EGFP signals after
transfecting pcDNA3.1-6xHis-T7-sRAGE-IntN (Figure 31). The immunocytochemistry
(ICC) staining further showed the cytoplasmic location of sRAGE-IntN protein in
HEK293T (Figure 32). Together, this result suggested the proper folding and
cytoplasmic location of 6xHis-T7-sRAGE-IntN protein in HEK293T.
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/31.avif)

▲ Figure 31: The fluorescence image of 6xHis-T7-sRAGE-IntN protein expression in HEK293T cells.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/32.avif)

▲ Figure 32: Immunocytochemistry analysis of 6xHis-T7-sRAGE-IntN protein in 293T cells.

We then performed the Western Blotting (WB) to examine whether the 6xHis-T7-sRAGE-IntN
protein was secreted into culture medium. The WB result indicated that the
6xHis-T7-sRAGE-IntN remained in the intracellular soluble fraction and was not
secreted into the culture medium (Figure 33).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/33.avif)

▲ Figure 33: Western blot analysis of 6xHis-T7-sRAGE-IntN expression in HEK293T cells. C: control; T: transfection; S: soluble supernatant; P: insoluble pellet. (A) cell medium (B) cell lysate.

**Constructing the pcDNA3.1-sRAGE-IntN, pcDNA3.1-mut-sRAGE-IntN, pcDNA3.1-IntC-6xHis-CBD, and pcDNA3.1-6xHis-IntC-CBD**

To build the correct construct without T7-tag inside, we re-cloned the gene blocks of
sRAGE-IntN, mut-sRAGE-IntN, IntC-6xHis-CBD and 6xHis-IntC-CBD into pcDNA3.1 using
HindIII and EcoRI. The sequence was verified by Sanger sequencing before transfection
into HEK293T for protein expression (Figures 34-37).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/34.avif)

▲ Figure 34: The electrogram shows the boundary between pcDNA3.1 and sRAGE-IntN.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/35.avif)

▲ Figure 35: The electrogram shows the boundary between pcDNA3.1 and mut-sRAGE-IntN.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/36.avif)

▲ Figure 36: The electrogram shows the boundary between pcDNA3.1 and IntC-6xHis-CBD.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/37.avif)

▲ Figure 37: The electrogram shows the boundary between pcDNA3.1 and 6xHis-IntC-CBD.

**Expression of sRAGE-IntN and mut-sRAGE-IntN in HEK293T**

To examine whether the sRAGE-IntN and mut-sRAGE-IntN protein could properly expressed
in HEK293T, we first applied fluorescence microscope to observe the EGFP signal in
transfected cells. The result show positive EGFP signal in either
pcDNA3.1-sRAGE-IntN or pcDNA3.1-mut-sRAGE-IntN transfected cells, suggesting
the successful expression of sRAGE-IntN and mut-sRAGE-IntN protein (Figure 38).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/38.avif)

▲ Figure 38: The fluorescence image of (A) sRAGE-IntN and (B) mut-sRAGE-IntN expression in HEK293T cells.

**Detecting and purifying of sRAGE-IntN and mut-sRAGE-IntN in HEK293T**

To confirm the sRAGE-IntN and mut-sRAGE-IntN secretion in culture medium, we performed
Immuno-blotting using an anti-His antibody. The result indicate the presence of
sRAGE-IntN and mut-sRAGE-IntN protein in both cell culture medium and the cell
lysate (Figure 39).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/39a.avif)

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/39b.avif)

▲ Figure 39: The Western blot analysis of (A) sRAGE-IntN and (B) mut-sRAGE-IntN in HEK293T. sup: supernatant

We then performed the protein purification using Ni-IDA resin to capture His-tagged
proteins. The result indicated that Non-specific proteins were effectively removed
by washing with PBS containing 600 mM imidazole. Both sRAGE–IntN and mut-sRAGE–IntN
were efficiently retained on the Ni-IDA resin, but could not be eluted and remained
bound to the resin (Figures 40 and 41).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/40.avif)

▲ Figure 40: Western blot analysis of transfected sRAGE-IntN into HEK293T cells and the purified result using an anti-His antibody.

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/41.avif)

▲ Figure 41: Western blot analysis of transfected mut-sRAGE-IntN into HEK293T cells and the purified result using an anti-His antibody.

**Detecting protein expression of IntC-6xHis-CBD and 6xHis-IntC-CBD in HEK293T**

Similarly, Western blot analysis was conducted to examine the expression of
IntC-6xHis-CBD and 6xHis-IntC-CBD in HEK293T cells. However, no band was observed at
the expected molecular weight of 12.65 kDa (Figure 42), suggesting that transfection
or protein expression was unsuccessful. Due to time constraints, successful
expression could not be demonstrated in this study. Future work will focus on
optimizing experimental conditions, such as adjusting transfection parameters, to
enhance protein expression efficiency.
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/42.avif)

▲ Figure 42: Western blot analysis of transfected IntC-6xHis-CBD and 6xHis-IntC-CBD into HEK293T cells. 8: IntC-6xHis-CBD 9: 6xHis-IntC-CBD

The sRAGE–IntN expressed in HEK293T cells could not be successfully purified,
further confirming that the presence of the Npu-DnaE intein interferes with elution
and purification. Once IntC-6xHis-CBD and 6xHis-IntC-CBD are successfully expressed,
we will proceed with purification experiments to further validate this observation.
After completing the purification process, intein-mediated splicing experiments will
be conducted to generate additional target fusion proteins and evaluate the overall
efficiency of the system.

## Evaluation of CBD anchorage on hydrogel

## Overview

To independently assess the binding capability of CBD to the hydrogel
matrix without interference from other structural domains, such as sRAGE or
intein, we constructed EGFP–CBD fusion protein to employ as a fluorescent reporter
probe. This construct served as an intein-independent validation module to
confirm the intrinsic collagen-binding functionality of CBD prior to its
incorporation into the full sRAGE–CBD fusion system.

## Design of the EGFP–CBD construct

To verify the hydrogel-binding capability of CBD in isolation, EGFP was genetically
linked to CBD via a flexible GGGSGGEF linker (EGFP-linker-CBD) to preserve the
structural integrity of CBD and minimize steric hindrance arising from
protein–protein interactions.
For *E. coli* expression, the native 6xHis tag and thrombin cleavage site present in the
pET15b vector were retained (6xHis-Thrombin-EGFP-linker-CBD). The 6xHis tag enabled
affinity purification, while the thrombin cleavage site allowed post-purification
removal of the tag to eliminate potential interference with CBD–hydrogel
interactions. In contrast, for HEK293T expression, only a 6xHis tag was
incorporated without the addition of a thrombin cleavage site (EGFP-6xHis-linker-CBD)
, thereby providing a simplified construct optimized for mammalian protein
expression.
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/new-result-43.avif)

▲ Figure 43: Gene block design of EGFP–CBD (A) for the *E. coli* expression and (B) for HEK293T expression.

## Expressing EGFP-CBD protein for hydrogel attachment test

**The confirmation of correct insertion of designed gene blocks in pET15b**

To express the 6xHis-Thrombin-EGFP-linker-CBD (named EGFP-CBD for short) protein in
*E. coli*, we ordered the codon-optimized gene blocks from IDT and performed Gibson
assembly to insert the gene blocks into pET15b vector. The sequence was verified
by Sanger sequencing before transformation into BL21 for protein expression (Figure
44).
![](http://static.igem.wiki/teams/5854/img/page-img/result/protein2/44.avif)

▲ Figure 44: The electrogram shows the boundary between pET15b and EGFP-CBD.

**The expression of EGFP-CBD protein in *E. coli***

We first induced the expression of EGFP-CBD with 1 mM IPTG at 37 °C and collected
samples at different time points (0, 2, 4, and 6 hr) (Figure 45A). However, the
expressed protein mainly accumulated in the inclusion bodies. To improve solubility,
we lowered the induction temperature to 20 °C and repeated the expression under the
same conditions. Despite this adjustment, EGFP-CBD was still found in the insoluble
fraction, indicating that it remained localized in inclusion bodies (Figure 45B).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/45a.avif)

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/45b.avif)

▲ Figure 45: The Coomassie blue staining of SDS-PAGE analysis of EGFP-CBD. SDS-PAGE analysis showed that EGFP-CBD remained insoluble at both 20 °C and 37 °C, and induction at 20 °C did not result in a notable increase in protein expression. S: supernatant; P: cell pellet. (A) Using 1mM IPTG to induced at 0, 2, 4, 6hr at 37℃. (B) Using 1mM IPTG to induced at 0, 2, 4, 6hr at 20℃.

**Solubilizing EGFP-CBD protein from inclusion body by 8M urea**

To solubilize the insoluble EGFP-CBD protein for subsequent experiments, we
applied 8 M urea extraction buffer to the insoluble fraction and confirmed efficient
extraction (Figure 46A). However, treatment with 8 M urea caused the EGFP to lose its
green fluorescence (Figures 46B and 46C).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/46a.avif)

![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/46bc.avif)

▲ Figure 46: (A) The Coomassie blue staining of SDS-PAGE analysis of EGFP-CBD extracted from the inclusion body. (B) The supernatant of EGFP-CBD lysed with 8 M urea was exposed to UV light. Using EGFP-4A-C7 (from 2023 CCU-iGEM) lysed with PBS + 0.1% lysis buffer as an example, functional EGFP would emit green fluorescence. (C) The pellet from EGFP-CBD lysed with PBS + 0.5% Triton X-100 showed visible green fluorescence to the naked eye. A 4x sample buffer was added to create a dark background in the microtube, facilitating fluorescence observation.

**Switching the expression system in mammalian cells**

To solve the problem of inclusion body expression of designed protein in *E. coli*,
we decided to shift the expression system to mammalian cell line HEK293T. We design
the new construct with codon-optimized for mammalian cells and cloned the EGFP-CBD
into pcDNA3.1 using HindIII and EcoRI. The sequence was verified by Sanger
sequencing before transfection into HEK293T for protein expression (Figure 47).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/47.avif)

▲ Figure 47: The electrogram shows the boundary between pcDNA3.1 and EGFP-CBD.

Then, we transfected the EGFP-CBD into HEK293T and verify the successful transfection
and expression result with fluorescence microscope (Figure 48), showing the EGFP
signals is right where inside the cell.
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/48.avif)

▲ Figure 48: The fluorescence image of EGFP-CBD protein expression in HEK293T cells.

Finally, Western blot analysis was performed to verify the size of the expressed
protein, confirming the presence of the intact EGFP-CBD fusion in the soluble
supernatant of cell lysate (Figure 49).
![](https://static.igem.wiki/teams/5854/img/page-img/result/protein2/49.avif)

▲ Figure 49: The Western blot analysis of EGFP-CBD in HEK293T.

Due to time constraints, we were unable to present the purification results of
EGFP–CBD. In future work, once the hydrogel component is fully developed, we will
combine the purified EGFP–CBD with the hydrogel to evaluate the binding capability
of the CBD domain to our collagen–chitosan hydrogel system.

## Future Work

After checking the expression of EGFP-CBD, we will perform Ni-IDA resin and apply it
to collagen-based hydrogel patches and assess attachment using EGFP fluorescence.
This will allow us to evaluate the collagen-binding capability of the CBD domain
independently of other protein modules.

Next, the final sRAGE–CBD fusion protein, assembled via intein-mediated ligation,
will be tested to confirm effective hydrogel anchoring. The functional performance
of the hydrogel-bound fusion protein will then be evaluated using ELISA to determine
its ability to capture AGEs. Together, these experiments will provide a stepwise
validation of both surface attachment and functional integrity before integration
into the full therapeutic system.

## Reference

1. Cheriyan, M., Pedamallu, C. S., Tori, K., & Perler, F. (2013). Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. *Journal of Biological Chemistry, 288*(9), 6202-6211.
2. Zettler, J., Schütz, V., & Mootz, H. D. (2009). The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. *FEBS letters, 583*(5), 909-914.

## Patch Layered Design

## Overview

In the AGE-Thwart, polyurethane (PU) was selected as the outermost protective
membrane due to its ability to provide reliable physical protection, preventing
external contamination and mechanical friction while reducing the risk of
exogenous infection.

For the inner layer contacting wound, collagen and chitosan were chosen.
Collagen mimics the extracellular matrix, promotes angiogenesis and collagen
deposition, and accelerates tissue regeneration (Cynthia Ann Fleck . 2010).
In addition, collagen serves as a suitable platform for anchoring sRAGE by
collagen binding domain. Chitosan, on the other hand, possesses inherent
antibacterial properties and excellent exudate absorption capacity (Ijaz Bano . 2017).

## Inspiration

Inspired by the work of the 2021 iGEM team MR.SA, the original design of patch
consisted of three distinct layers: a polyurethane protective layer, a chitosan
absorption layer, and a collagen contact layer.
![](https://static.igem.wiki/teams/5854/img/page-img/result/dl-result1.webp)
To optimize the patch, we explored the possibility to reduce the complexity
of original design. We found that, in article Effects of Chitosan-Collagen
Dressing on Wound Healing in Vitro and in Vivo Assays, dressings composed
of both collagen and chitosan demonstrate enhanced wound healing compared
to those made of collagen or chitosan alone (Min-Xia Zhang, 2021).

So, we redesigned the patch from a three-layered structure to a two-layered
structure, **integrating collagen and chitosan into a unified contact layer**.
![](https://static.igem.wiki/teams/5854/img/page-img/result/dl-result2.webp)

## The Preparation of Collagen-Chitosan Layer

## The effects of NaOH concentration in chitosan crosslinking

**Design:**

A uniform hydrogel of collagen and chitosan was prepared using a physical
crosslinking approach. Collagen and chitosan were individually dissolved in
acetic acid and subsequently mixed at a defined ratio, which was then
neutralized to pH 7 by 1M NaOH, followed by freeze-drying to obtain hydrogel
(Pablo Sánchez-Cid .2022).

In practice, we found that the crosslink of collagen and chitosan mixture by
1M NaOH neutralization cause a sudden pH change to alkaline state,
hampering the hydrogel formation. Using NaOH with low concentration partially
solves this problem, but increases the final volume of reaction. Since the
small volume of crosslink reaction makes it not easy to be measured by
convectional pH meter, we decide to perform an acid-base titration modeling
to determine the suitable NaOH concentration for crosslink (Figure 1).
![](https://static.igem.wiki/teams/5854/img/page-img/result/figure-1.webp)

▲ Figure 1: Three titration curves of acetic acid using (A)0.01 M, (B)0.1 M and (C)1 M NaOH. Volume of (D)0.01 M, (E)0.1 M and (F)1 M NaOH required to reach the equivalence point. (G)line represents the pH of acetic acid at the equivalence point.

Based on modeling result, the following procedure was carried out.
Firstly, 775ul of 1% chitosan was added separately to three 6-cm dishes.
Secondly, 73.42ul 1M, 734.2ul 0.1M and 7342ul 0.01M NaOH was added to
three dishes to raise pH to 6. Lastly, these two dishes were lyophilized.

**Result and conclusion:**

The chitosan crosslinked with 0.01 M NaOH showed a uniform and intact sheet.
Other concentrations, 0.1M and 1M produced incomplete sheets and the
structure of these sheets were not uniform (Figure 2).
![](https://static.igem.wiki/teams/5854/img/page-img/result/figure-2.webp)

▲ Figure 2: The chitosan solution crosslinked by (A) 0.01M, (B) 0.1M, and (C) 1M NaOH after free-drying in 6-cm dishes.

To sum up, we selected 0.01M NaOH to is crosslink the chitosan and collagen mixture.

## 0.01M NaOH is not suitbale for Collagen and Chitosan Hydrogel crosslink

**Design:**

After determining the NaOH concentration in crosslinking, we first
apply the 98.421ul 0.01M NaOH to neutralize and crosslink the
2.5ml 3.1%(mg/ml) collagen and 775ul 1%(g/ml) chitosan mixture (1:1).

**Result and conclusion:**

After freeze-drying, the hydrogel showed a web-like structure rather than forming an intact sheet (figure 3).
![](https://static.igem.wiki/teams/5854/img/page-img/result/figure-3.webp)

▲ Figure 3

We speculated that the reason for the hydrogel forming a web-like structure
is the excess amount of NaOH in neutralizing collagen in HCl solution.
The base area of container may also cause the uneven distribution of solution.

## Adjusting the NaOH concentration and container base area

**Design:**

We transferred the contents of the previous experiment from 6 cm dishes to 3.5 cm
dishes. In the first step, we added 775ul of 1% chitosan separately to two
3.5-cm dishes. Secondly,73.42ul 1M and 734.2ul 0.1 M NaOH was added to two
dishes to crosslink chitosan. Lastly, these two dishes were lyophilized.

**Result and conclusion:**

The hydrogel prepared with 0.1 M NaOH formed an intact sheet, whereas the hydrogel prepared with 1 M NaOH did not (Figure 4).
![](https://static.igem.wiki/teams/5854/img/page-img/result/figure-4.webp)

▲ Figure 4: figure of hydrogel crosslinked by (A)1M NaOH and(B) 0.1M NaOH after free-drying in 3.5 cm dishes.

The result showed that 0.1M NaOH was able to form a complete hydrogel simply by changing the container.

## Applying the new condition to Collagen–Chitosan Hydrogel crosslink

**Design:**

Due to the running out of collagen, we only have collagen (Enzo Life Sciences, USA)
with low concentration (100ug/ml) for testing. We first lyophilized 15 mL of collagen
and resolve into 1.5 ml of acetic acid (1 mg/ml). The collagen solution was then mixed
with 0.15ml chitosan solution and stirred for 1 hour, then transferred into 3.5 cm
dishes. Second, 1.56315mL of 0.1 M NaOH was added to complete the crosslinking.
Finally, the hydrogels were lyophilized.

**Result and conclusion:**

The resulting hydrogel formed a thin film; however, the edges of the film
were slightly damaged (figure 5).
![](https://static.igem.wiki/teams/5854/img/page-img/result/figure-5-new.avif)

▲ Figure 5

In our opinion, this hydrogel was better than the previous one with
collagen and chitosan due to our adjustment in concentration of NaOH and
container size. However, the hydrogel didn’t form an intact sheet. We
thought this was due to the total content of collagen and chitosan being
too low. So, we will try to increase the concentration of collagen
solutions in the future.

## Future Vision

In future work, we plan to increase collagen concentration to fabricate a 1:1
hydrogel. After successfully producing the 1:1 hydrogel, we will adjust the
collagen-to-chitosan ratio and conduct detailed structural and antibacterial
evaluations to further optimize the patch composition. Scanning Electron
Microscopy (SEM) will be utilized to observe the microstructure and quantify
the pore characteristics under different collagen-to-chitosan ratios. These
analyses will provide insights into how the matrix architecture affects protein
immobilization and wound healing performance.

Furthermore, agar diffusion assays will be performed to systematically evaluate
the antibacterial efficacy of the hydrogels against common wound pathogens.
By correlating the SEM findings with antibacterial assay results, we aim to
determine the optimal material ratio that achieves a balance between protein
immobilization, antibacterial activity, and biocompatibility.

## Reference

1. Fleck, C. A. (2010). Modern collagen wound dressings: Function and purpose. *Advanced Drug Delivery Reviews, 62*(4–5), 205–219.
2. Bano, I. (2017). Chitosan: A potential biopolymer for wound management. *International Journal of Biological Macromolecules*. Advance online publication.
3. Zhang, M.-X., Zhao, W.-Y., Fang, Q.-Q., et al. (2021). Effects of chitosan-collagen dressing on wound healing in vitro and in vivo assays. *Journal of Applied Biomaterials & Functional Materials, 47*(4).
4. Sánchez-Cid, P., Jiménez-Rosado, M., Rubio-Valle, J. F., Romero, A., Ostos, F. J., Rafii-El-Idrissi Benhnia, M., & Perez-Puyana, V. (2022). Biocompatible and thermoresistant hydrogels based on collagen and chitosan. *Polymers, 14*(2), 272.
5. Sánchez-Cid, P., Jiménez-Rosado, M., Alonso-González, M., Romero, A., & Perez-Puyana, V. (2021). Applied Rheology as Tool for the Assessment of Chitosan Hydrogels for Regenerative Medicine. *Polymers, 13*(13), 2189.
6. Sánchez-Cid, P., Jiménez-Rosado, M., Alonso-González, M., Romero, A., & Pérez-Puyana, V. (2020). Fabrication and characterization of hydrogels based on gelatinised collagen with potential application in tissue engineering. *Polymers, 12*(5), 1146.

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