iGEM 2022 — Team edinburgh-uhas-ghana

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Contents

* [1) July](#1)
* [2) August](#2)
* [3) September](#3)
* [4) October](#4)

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Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)

# July

### Week 1 (July 4 – 10)

We prepared stock solutions of MgCl2, CaCl2 and glycerol. We produced overnight cultures of J23100, B0034, L2U2H09, Uac level 0 acceptor and LacZ level 1 acceptor, and then miniprepped them. We also prepared a stock of competent TOP10 *E. coli* cells, and stored them at -80°C. We also attempted to use PCR to add JUMP fusion sites to a MerR part, while removing the stop codon. Unfortunately, this did not work.

### Week 2 (July 11 – 17)

We domesticated mut\_MerR, PbrR, and ArsR, but were unable to transform them into cells as our GMRA had not been approved yet. In the meantime, we did a bradford calibration curve.

### Week 3 (July 18 – 24)

We started PCR of silica tags for PET immobilization, which initially failed due to primer dimers, but then succeeded when we slightly raised the temperature. Our iSpinach biosensor constructs had arrived as well, so we started a PCR of them, which failed a few times and eventually succeeded after enough optimization. We also produced a stock solution of carboxymethycellulose (CMC) at 3% w/v.

### Week 4 (July 25 – 31)

Dr. Marcos Valenzuela-Ortega gave us a stock of cells with a MerR O part plasmid after our PCR amplifications failed. We produced an overnight culture of these cells, and miniprepped the plasmids out. We prepared a stock of competent SHuffle *E. coli* cells. We also began level 1 assembly of PET biodegradation parts. We also transformed the level 1 assembly products into TOP10 cells, plated on IPTG, Xgal, and Kanamycin plates. We made CMC hydrogels, as well as CMC/CA (citric acid) hydrogels. We observed that the CMC hydrogel had grid-like non-uniform square shapes, while the CMC-CA hydrogel was a hard uniform thin layer. We were able to use the 2021 Edinburgh iGEM Team’s GMRA for PETase related transformations, but all biosensor related transformations still needed to wait for GMRA approval.

# August

### Week 5 (August 1 – 7)

We miniprepped the level 1 PET biodegradation assemblies out of TOP10 and transformed SHuffle with them. We performed a swell test of both hydrogels, and observed that the CMC hydrogel flaked easily, while the CMC-CA hydrogel became a syrup-like gel and absorbed the water very well. Our bioremediation parts finally arrived, so we began domesticating them. We also did a level 1 JUMP assembly of the untagged sfGFP control.

### Week 6 (August 8 – 14)

We did domestications of our new tags; the His-, SUMO-, CBD-, and SB7 Si-tags. Finally, our GMRA was approved, so we could transform our biosensor assemblies for expressing transcription factors. We then prepared lysates of our cells, and ran them on an SDS-PAGE. We performed colony PCRs for all our PET biodegradation related parts, and most came out as a success. We did another assembly of all the failed assemblies and transformed them into TOP10.

### Week 7 (August 15 – 21)

We performed all Bioremediation and Directed Evolution related level 1 assemblies, but they all failed, so we began troubleshooting. We spent all week troubleshooting this, and found out a batch of our competent BL21(DE3) cells was at fault. We also domesticated SELIS parts for directed evolution. We began several trials of in vitro transcription. We observed an anomalous result which exhibited a color change, but no fluorescence.

### Week 8 (August 22 – 28)

While troubleshooting the biosensors, we found out we had used 10,000x more lysozyme than needed while making the lysates, so we began making them again with the correct amount. We retransformed cells with the Bioremediation level 1 assemblies, and got some white colonies. We did colony PCR of these colonies, which yielded lanes with no bands. We tried this again and got the same result, and found that none of them had the insert we were looking for. We began troubleshooting the assemblies and transformations again.

# September

### Week 9 (August 29 – September 4)

We realized there was an issue with the strain we were using to express the transcription factors for biosensing, as we were trying to use TOP10 for protein expression. We miniprepped the constructs out of TOP10, and transformed into BL21(DE3). We also did error-prone PCR of our transcription factors and metallothioneins for directed evolution. We also retried a few level 1 assemblies and transformations under different conditions, and they were a success. We decided to break down the remaining level 1 assemblies into four batches, to prevent overusing consumables on batches that would fail, and did the first of four batches. We also did the first PETase immobilisations and began testing PETase activity.

### Week 10 (September 5 – 11)

This week, we needed to move labs, and the new lab had more restrictions on the number of people we could have in. We prepared lysate of the transcription factor expression cells, made new aliquots of competent BL21(DE3) cells, did PETase induction from SHuffle liquid culture and did the second and third batches of level 1 assemblies. We also prepared plates with AgNO3 for directed evolution, and made level 1 assemblies of all error prone metallothioneins except *P. fluorescens* and *C. sapidus*. We removed the stop codon from MHETase using PCR. We transformed the first batch into BL21(DE3) cells, sonicated them, and checked for batch 1 protein expression with SDS-PAGE. We continued trying different variations of biosensor trials, and ordered in new constructs expressing different aptamers.

### Week 11 (September 12 – 18)

We did more sonications of cells expressing PETases, and finished the activity assays. We did HPLC for fast PETases, redid the third batch of assemblies and did the fourth batch and transformed them into TOP10, did colony PCR of the second batch. We transformed the second batch into BL21(DE3) cells, sonicated them, and checked for protein expression with SDS-PAGE. We tried more biosensor trials with the new constructs, but we could still not get any fluorescence results. We plated the error-prone PCR metallothionein expression constructs on the AgNO3 plates.

### Week 12 (September 19 – 25)

We had organized the batches into order of priority, and as we had dropped a few experiments, there were quite a few assemblies in the third and fourth batches we didn’t need anymore. For those we did need, we transformed them into BL21(DE3) cells, produced lysate and checked for protein expression. We immobilized our CBD-tagged proteins on the cellulose hydrogels and baked them to bind them. We did HPLC of MHETase. We continued troubleshooting the biosensors.

# October

### Week 13 (September 26 – October 2)

We performed the N- and C-filler characterization experiment. We prepared and submitted hydrogel samples mixed with solutions of heavy metals for native-MS and ICP-MS.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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Contents

* [1) Biosensing](#1)
  + [1.1) RNA aptamer-fluorophore interaction](#1.1)
    - [1.1.1) Cycle 1](#1.1.1)
    - [1.1.2) Cycle 2](#1.1.2)
  + [1.1) Transcription factor engineering](#1.2)
    - [1.1.1) Cycle 1](#1.2.1)
    - [1.1.2) Cycle 2](#1.2.2)
* [2) Bioremediation](#2)
  + [2.1) Cycle 1](#2.1)
  + [2.2) Cycle 2](#2.2)
  + [2.3) Cycle 3](#2.3)
  + [2.4) Cycle 4](#2.4)
* [3) Bioconversion](#3)
  + [3.1) Cycle 1](#3.1)
  + [3.2) Cycle 2](#3.2)

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# Biosensing

### RNA aptamer-fluorophore interaction

##### Cycle 1 - Making Spinach2 biosensors for cadmium, mercury, lead and arsenic

**Design:**

RNA of Spinach2 forms an aptamer that can bind certain fluorophores, such as DFHBI, and cause fluorescence. This allows for creating a cell-free system by using a polymerase to transcribe the DNA sequence, making RNA and thus causing fluorescence. DNA sequences incorporating a T7 promoter, the binding site for the specific transcription factor and the aptamer-yielding sequence flanked by F30 sites were constructed.

**Build:**

The sequences were ordered, resuspended in TE buffer, and multiplied by PCR as outlined in the experimental section. After the PCR reaction, the DNA concentration was quantified using NanoDrop and the construct sizes were verified using agarose gel electrophoresis.

**Test:**

The PCR mixtures were used to run a fluorescence test as outlined in the experimental section, by allowing the DNA in the PCR mixture to be transcribed by T7 polymerase to produce and RNA aptamer capable of binding to DFHBI, causing fluorescence.

**Learn:**

No fluorescence was observed from the tests. The lack of fluorescence could have been caused by the poor quality of DNA, as no PCR clean-up was performed before use in the reaction and DNA quality is of utmost importance in cell-free transcription systems. Another cause for the lack of fluorescence may have been that the combination of fluorophore and aptamer was suboptimal. As such, a different set of RNA aptamer constructs using iSpinach, Broccoli and Squash were ordered to test.

##### Cycle 2 - Making iSpinach, Broccoli and Squash biosensors for cadmium, mercury, lead and arsenic

**Design:**

The rationale behind the DNA sequences was identical to the one used for Spinach2, with the aptamer being replaced by iSpinach, Broccoli or Squash.

**Build:**

The sequences were ordered, resuspended in TE buffer, and multiplied by PCR as outlined in the experimental section. After the PCR reaction, the presence of DNA and construct sizes were verified using agarose gel electrophoresis. PCR clean-up was performed, and the purified DNA concentrations were quantified using NanoDrop.

**Test:**

The PCR mixtures were used to run a fluorescence test as outlined in the experimental section, by allowing the DNA in the PCR mixture to be transcribed by T7 polymerase to produce and RNA aptamer capable of binding to DFHBI, causing fluorescence. After checking for fluorescence, the reaction mixture was run on an agarose gel to observe RNA smears indicating transcription.

**Learn:**

Neither fluorescence nor RNA transcription was observed. This indicates that either the reaction conditions were not conducive to T7 transcription activity or the that the polymerase could not transcribe these aptamers either.

### Transcription factor engineering

##### Cycle 1 - Expressing transcription factors for mercury, lead, cadmium, and arsenic in *E. coli* TOP10

**Design:**

ArsR, PbrR, MerR and mut\_MerR are transcription factors (TFs) that can bind DNA and arsenic, lead, mercury or cadmium respectively. When no metal is present, the TFs can bind to specific DNA sites, preventing any enzymes from moving along the sequence. However, when bound to a metal the TFs unbind the DNA. As such, transcription can be controlled depending on the presence of heavy metals in the reaction mixture.

**Build:**

The sequences were assembled using JUMP assembly as outlined in the experimental section. Successful assemblies were verified using the colour of the colony. Colonies containing level 0 parts with the insert of interest, while colonies without the insert were green as they retained GFP. Successful level 1 assemblies were white as the insert of interest replaced the lac operon, while colonies without the interest were blue as they had the lac operon while being grown on agar containing Xgal and IPTG. The presence of the insert of interest in the assembly was further verified by agarose gel electrophoresis of restriction digests of the miniprepped level 1 plasmids.

**Test:**

The TOP10 strain of *E. coli* was transformed with the level 1 plasmids of the TFs and a culture was grown. Protein expression was induced by growing at 20 °C for 2 hours, the cells were then lysed according to the protocol in the experimental section. 2 mg of the lysate was run on an SDS-PAGE gel for qualitative analysis.

**Learn:**

No band corresponding to the TFs was present in the gel, thus no expression of transcription factors was present. As such, instead of *E. coli* TOP10, a strain optimised for protein expression called *E. coli* BL21(DE3) was to be used.

##### Cycle 2 - Improving expression of transcription factors in *E. coli*

**Design:**

ArsR, PbrR and MerR were used for further testing, with the rationale behind it staying the same.

**Build:**

The sequences were assembled using JUMP assembly as outlined in the experimental section. Successful assemblies were verified using the colour of the colony. Colonies containing level 0 parts with the insert of interest, while colonies without the insert were green as they retained GFP. Successful level 1 assemblies were white as the insert of interest replaced the lac operon, while colonies without the interest were blue as they had the lac operon while being grown on agar containing Xgal and IPTG. The presence of the insert of interest in the assembly was further verified by colony PCR of white colonies, performed as written in the experimental section.

**Test:**

The BL21(DE3) strain of *E. coli* was transformed with the level 1 plasmids of the TFs and a culture was grown. Protein expression was induced by growing at 20 °C for 2 hours, the cells were then lysed according to the protocol in the experimental section. 3 mg of the lysate was run on an SDS-PAGE gel for qualitative analysis.

**Learn:**

Only ArsR and PbrR level 1 assemblies could be successfully made, however, both had a band corresponding to the size of their respective TFs, indicating that protein expression occurred.

# Bioremediation

### Metallothionein-Displaying 3C Hydrogel

##### Cycle 1 - Making Citric-acid-crosslinked carboxymethylcellulose (CMC-CA) hydrogels

**Design:**

The standard properties of biopolymer-based hydrogels include insolubility in water, swelling and degradation after use. We determined the sizes and compositions of CMC and citric acid and the suitable conditions for making CMC-CA hydrogels that fulfill these criteria.

**Build:**

We chose 3% w/v CMC and 15% w/v citric acid as the composition of our hydrogels and made a solution of them. We poured the solutions into 2 kinds of molds: 25 ml frog-shaped silicon molds and 1 ml box-shaped silicon molds. They were dehydrated in the oven at 40 degrees overnight. Afterwards, we increased the oven temperature to 80 degrees and left the hydrogel samples there overnight to allow optimal conditions for crosslinking.

**Test:**

The properties of the resulting CMC-CA hydrogels were investigated by swelling and degradation tests. For the swelling test, the CMC-CA hydrogels were submerged in water for 1 hour, and their initial and final masses were compared. The degradation test was carried out after the swelling test by dehydrating the CMC-CA hydrogels in the oven at 40 degrees overnight, and their initial (original mass before swelling tests) and final masses were compared.

Additionally, we tested the amount of protein our CMC-CA hydrogel can absorb. We incubated a fragment of the hydrogel in a BL21(DE3) lysate in 1 hour, then compared the total protein concentration of the initial lysate and that of the supernatant after addition of the hydrogel (using a standard Bradford assay).

**Learn:**

* The large frog-shaped hydrogels swelled by 32.63% of their initial mass and after re-dehydration, degraded by 76.4% of their initial mass.* The small box-shaped hydrogels did not swell significantly. In fact, two out of three hydrogels in our triplicated experiment shrunk a bit (average swelling degree was –2.045%). This may be because using smaller volumes made the hydrogels thinner and more fragile (easy to get fragmented when handled). After re-dehydration, these hydrogels degraded by 77.825% of their initial mass.
  * 20 mg of our CMC-CA hydrogel absorbs 52.62% of proteins from 1 ml of a lysate of 1000 mg/ml total protein concentration.

Overall, our CMC-CA hydrogels fulfilled the standard properties of typical biopolymer-based hydrogels and are highly absorbent towards proteins in general. The size of the hydrogels may significantly affect their swelling degree.

##### Cycle 2 - CMC-CA hydrogels versus silica beads for protein immobilisation

**Design:**

We need to test for selective immobilisation of CBD-tagged proteins to the CMC-CA hydrogels by fusing it to an indicator, such as sfGFP. We designed the composite part CBD-sfGFP for producing N-terminal CBD-tagged sfGFP.

We also investigate how effective protein immobilisation on our CMC-CA hydrogels is by comparing it with other solid-phase materials as we were unsure how effective hydrogels are. We decided to explore silica beads as the alternative platform for protein immobilisation. We designed the composite part SB7-sfGFP for producing sfGFP with an N-terminal silica-binding peptide. SB7 denotes a short arginine-rich sequence designed for silica bead affinity tagging.

**Build:**

The JUMP plasmid containing our 6xHis-CBD-sfGFP level 1 insert was cloned in TOP10, then transformed into and expressed in BL21(DE3). The culture was lysed, and a 20-30 mg fragment of our small box-shaped CMC-CA hydrogel was incubated in the lysate for 1 hour.A BL21(DE3) lysate of SB7-sfGFP was obtained using the same method mentioned above. 20 mg of Celite545 silica beads were incubated in the lysate for 1 hour.

**Test:**

The sfGFP fluorescence intensities of the initial lysates and supernatants after incubation of the hydrogel and silica beads were measured using a microplate reader. The change in fluorescence intensity indicates the proportion of recombinant sfGFP immobilised to the solid platforms pelleted and separated from the supernatant. For verifying the test, the experiment was replicated using the control lysate containing sfGFP without CBD fusion.

**Learn:**

There was no reduction in fluorescence intensity for the SB7-sfGFP control samples, suggesting that the SB7 peptide has little to no affinity for silica beads.

The fluorescence intensities for both CBD-sfGFP and the non-tagged sfGFP control samples dropped drastically, indicating that the CMC-CA hydrogels capture proteins non-selectively which further reinforces the results of our protein absorption test from our previous engineering cycle. The initial fluorescence intensity of the lysate containing CBD-sfGFP was significantly lower than that of the lysate containing the non-tagged sfGFP control, indicating that CBD-fusion may have a suppressive effect on either sfGFP expression in *E. coli* or its fluorescence.

Therefore, our CMC-CA hydrogels combined with CBD-tagged proteins (3C hydrogels) are a much more effective method for immobilising proteins compared to displaying silica-affinity-tagged proteins on silica beads.

However, although our hydrogels significantly trapped the CBD-sfGFP, this test still does not clearly show whether the cellulose hydrogel matrix has a significant selective affinity for the CBD compared to other proteins.

##### Cycle 3 - Improved verification of selective immobilisation of CBD-tagged proteins

**Design:**

Because the superabsorbent property of our CMC-CA hydrogels makes it difficult to determine whether cellulose-based solid materials selectively interact with CBD-tagged proteins compared to other compounds in the lysates, we decided to substitute our CMC-CA hydrogels with another cellulose-based material as an attempt to improve our CBD-cellulose binding assay. We still need a visible indicator for this, so we used the same CBD-sfGFP fusion protein from the previous test.

**Build:**

20 mg of Avicel insoluble microcrystalline cellulose was incubated in the BL21(DE3) lysate containing our CBD-sfGFP for 1 hour.

**Test:**

The sfGFP fluorescence intensities of the initial lysate and the final supernatant after separating out the pelleted cellulose was measured using the microplate reader. We also visualised the cellulose pellet using a blue light box to confirm that the pellet contains the CBD-tagged sfGFP. For verifying the test, the experiment was replicated using the control lysate containing sfGFP without CBD fusion.

**Learn:**

There was only a slight, insignificant decrease in fluorescence intensity for the non-tagged sfGFP sample, indicating that some sfGFP may be physically trapped in the microcrystalline cellulose by chance. Meanwhile, there was a significant reduction in the fluorescence intensity of the CBD-sfGFP sample. Furthermore, we observed in the light box that the cellulose pellet after incubation with the CBD-sfGFP lysate displayed a significantly, visibly brighter green fluorescence than the pellet that was incubated with the non-tagged sfGFP lysate. Therefore, this test proved that cellulose-based solid materials selectively bind the CBD and so immobilise CBD-tagged proteins.

##### Cycle 4 - Testing metal capturing of metallothionein-displaying 3C hydrogels

**Design:**

Our final engineering cycle is the immobilisation of CBD-tagged metallothionein to our CMC-CA hydrogels. We designed the composite part CBD-MT for producing the CBD-metallothionein fusion proteins.

**Build:**

The JUMP plasmid containing our CBD-MT level 1 insert was cloned in TOP10, then transformed into and expressed in BL21(DE3). The culture was lysed, and fragments of our small box-shaped CMC-CA hydrogel were incubated in the lysate for 1 hour to decorate them with the CBD-tagged MT to complete the 3C hydrogel build.

After incubation, the hydrogels were washed to remove as much unbound proteins and lysis buffer components capable of metal chelation (e.g. EDTA and DTT) as possible. They were then separately incubated in Zn(II) (the native metal ion of metallothioneins in cells) or Ni(II) (a heavy metal ion) solutions for 1 hour. This experiment was replicated using a negative control BL21(DE3) lysate for verifying the results.

**Test:**

The initial (pre-hydrogel treatment) and final (post-hydrogel treatment) Zn(II) or Ni(II) concentrations were measured and compared with each other using inductively coupled plasma mass spectrometry (ICP-MS). The reduction in Zn(II) or Ni(II) concentration represents the amount of Zn(II) or Ni(II) captured by the lysate-decorated CMC-CA hydrogels.

**Learn:**

* Unexpectedly, the negative control hydrogels captured a slightly higher amount of Zn(II) (i.e. slightly larger Zn(II) concentration reduction in solution) than our metallothionein-displaying 3C hydrogels. A possible explanation for this is incubating absorbent hydrogels in lysates may have lead to non-selective immobilisation of many proteins, and there are many Zn(II)-binding proteins in cells other than metallothioneins. Another explanation is that the washing step may not be adequate for removing the lysis buffer components that include metal-chelating compounds EDTA and DTT.
* Our metallothionein-displaying 3C hydrogels captured a slightly higher amount of Ni(II) (i.e. slightly larger Ni(II) concentration reduction in solution) than the negative control hydrogels. We speculate that the Ni(II) binding capacity of our hydrogels after decoration with CBD-MT was improved by only a small margin due to low expression of CBD-MT in *E. coli* (thus, may be present at relatively low amounts in the lysates) which is a factor we were not able to measure due to time constraints.

Overall, our engineered 3C hydrogels have the potential to capture heavy metal ions to at least a higher extent compared to typical biopolymer-based hydrogels without immobilised metallothioneins, but further measures (especially using purified CBD-MT instead of its host *E. coli* lysate) could be taken to improve them.

# Bioconversion

### PETase and MHETase immobilization Engineering

##### Cycle 1 - Assessment of PETase and MHETase variants activity after fusing with silica tags

**Design:**

We wanted to assess the effect of silica tag fusion towards the PETase activity. Therefore, we designed a collection of fusion proteins with different combinations of functional enzymes and silica tags (Figure 1). The position of silica tags can be either N-terminal or C-terminal of the functional enzyme. Due to the presence of disulfide bond in both PETase and MHETase, we chose the *E. coli* SHuffle strain for protein expression. The codon optimization was done on Benchling for protein expression in *E. coli* (K12). After protein expression, we needed to assess the protein activity under 37°C, which is working temperature for our design.

![](https://static.igem.wiki/teams/4390/wiki/pet/pet-design-parts.jpeg)

**Figure 1.**The schematic representation of the composite part designed for PETase and MHETase immobilization. Dou-PETase: Double mutant PETase (S238F/ W159H); Tri-PETase: Triple mutant PETase (T140D/R224Q/N233K); FAST-PETase: Quintuple mutant PETase (S121E/D186H/R224Q/N233K/R280A). L2NC and Car9 were different silica tags.

**Build:**

Based on the modular rationale of JUMP assembly, we constructed 3 Dou-PETase plasmids, 6 Tri-PETase plasmids, 6 FAST-PETase plasmids and 3 MHETase plasmids. All plasmids were transformed into *E. coli* SHuffle separately, and the cells were sonicated after protein expression. We centrifuged the cell lysates and took the supernatants for further assessment.

**Test:**

The PETase activity will be assessed based on para-nitrophenol-butyrate (pNPB) assay, since pNPB can be hydrolysed by PETase into para-Nitrophenol with maximum absorbance at 415 nm (Pirillo, V, et al., 2021). Active PET enzymes can produce a yellow colour visible to the naked eye.

**Learn:**

All Dou-PETase Tri-PETase, FAST-PETase and MHETase fused with different silica tags were soluble and active, and they showed different levels of higher activity compared to existing part Dou-PETase. The [Car9-linker-Tri\_PETase]. However, we didn’t assess the MHETase activity in the first cycle due to multiple limitations, including not commercially available MHET substrate, the complexity of using HPLC or proton NMR.

We also learnt that we should add LacO sequence between T7 promoter and B0034 RBS for inducible protein expression. The N-terminal L2NC tag can’t be made due to wrong primer design to obtain it.

##### Cycle 2 - Assessment of PETase-silica tag fusion proteins: immobilization rate and activity

**Design:**

After making sure the PETase-silica tag fusion proteins were still active, we needed to measure the silica immobilization efficiency of different constructs and assess the immobilized enzyme activity.  This cycle was also crucial to prove that there is no 3-phase problem for immobilized PETase (3-phase problem: whether the enzyme immobilized on the silica beads would undergo a phase change that would prevent the reaction from taking place properly). For cycle 1, we learnt that the lack of LacO in the plasmid design resulted in lower PETase expression, since we can’t induce expression with IPTG. Therefore, we increased the protein loading from (100 µg protein sample/ 20mg silica beads) to (500 µg protein sample/ 20mg silica beads).

**Build:**

The purification and immobilization were done simultaneously by incubating each ml of soluble cell lysates (500 µg/ml) with 20 mg silica beads (Celite 545) for 30 minutes, at 4°C. After incubation, the mixture was centrifuged, and the supernatant was taking out to measure non-immobilized protein concentrations. The remaining silica beads were resuspended in 1 ml buffer (pH7.0, 45 mM Na2HPO4-HCl, 90 mM NaCl, and 10% (v/v) DMSO) for further activity assessment based on pNPB assay.

**Test:**

The initial protein concentration in the cell lysates and non-immobilized protein concentration after incubation were measured by Bradford assay with comparison to the standard curve. The immobilization efficiency was calculated. The immobilized PETase activity was measured with pNPB assay.

**Learn:**

* The [FAST\_PETase-linker-Car9] showed the highest immobilization efficiency over all the constructs (100%). And the [FAST\_PETase-linker-L2NC] showed the lowest immobilization efficiency (11.93%). The Tri-PETase with silica tag shows low immobilization efficiency comparing to the FAST\_PETase with silica tags after we corrected the data with empty control respectively.
* The [FAST\_PETase-linker-L2NC] shows the highest activity towards pNPB after immobilizing on the Celite545, and the fold change of activity over empty control increased from 2.79 to 16.62, but its immobilization rate was the lowest. Referring to the relatively low immobilization efficiency of [FAST\_PETase-linker-L2NC], we assumed that the protein loading on each silica bead should be low.

In this cycle, we first changed the immobilization protocol. The original protocol we followed was preparing the silica beads in the buffer. However, we can’t precisely take out the suspended silica beads from the solution. Therefore, we measured the weight of silica beads at first, and add the buffer and protein samples later to produce more consistent results.

Moreover, the lack of LacO can’t be simply and fully compensated by increasing the protein loading. We observed the binding of untagged PETase and proteins that constitutively expressed to the silica beads. And the inhibition effect brought by higher protein loading to the silica beads. We learnt that the protein loading per silica bead should be well defined to maintain the enzyme activity after immobilization.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

Contents

* [1) General Experiments](#1)
* [2) JUMP assembly](#2)
* [3) *In vitro* transcription](#3)
* [4) Protein Immobilisation](#4)

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# General Experiments

### Competent cell preparation

1. 1 ml of an overnight *E. coli* culture was transferred to 100 ml of LB media and incubated at 37 °C and 200 rpm until OD = 0.4 - 0.6.
2. The culture was poured into 50 ml falcon tubes, cooled on ice for 30 minutes and centrifuged at 4 °C and 4000 rpm for 5 minutes.
3. The media was discarded, and the pellet was resuspended in 25 ml 0.1 M of ice cold MgCl2.
4. The cells were cooled on ice for 30 minutes and spun down at 4 °C 4000 rpm for 5 minutes.
5. The supernatant was discarded, the pellet was resuspended in 25 ml 0.1 M of ice cold CaCl2.
6. The cells were cooled on ice for 30 minutes and spun down at 4 °C at 4000 rpm for 5 minutes.
7. The supernatant was discarded, and the pellet was resuspended in 1.25 ml ice cold CaCl2/glycerol solution (1.7 mL 0.1 M CaCl2, 0.3 ml 100 % glycerol).
8. The suspension was divided into 100 µl aliquots that were flash frozen in liquid nitrogen and stored at -80 °C.

### Minipreps

1. An overnight culture was grown and centrifuged at 4000 rpm for 5 minutes.
2. The media was removed, and the pellet resuspended in 250 µl P1 buffer.
3. 250 µl of P2 buffer were added, and the mixture was mixed well.
4. 350 µl of N3 buffer were added, and the mixture was mixed well.
5. The tube was centrifuged at 13000 rpm for 10 minutes, 800 µl of the supernatant were added to the QIAprep spin column.
6. The column was centrifuged at 13000 rpm for 1 minute.
7. 0.75 ml PE buffer were added to the column.
8. The tube was centrifuged at 13000 rpm for 1 minute.
9. The column was transferred to a new Eppendorf tube, 50 µl of EB buffer were added to the column.
10. DNA was eluted after centrifuging 13000 rpm for 1 minute.

### PCR cleanup

1. 6X PB buffer was added to the PCR mixture and moved to the QIAquick spin column.
2. The column was centrifuged at 13000 rpm for 1 minute, the flow through was discarded.
3. 0.75 ml PE buffer were added to the column.
4. The column was centrifuged again at 13000 rpm for 1 minute, the flow through was discarded.
5. The column was centrifuged at 13000 rpm for 1 minute to remove any residual buffer.
6. The column was transferred to a new Eppendorf tube and 30 µl of EB buffer was added.
7. Purified DNA was eluted by centrifuging the column at 13000 rpm for 1 minute.

### Transformations

1. 10 µl of plasmid DNA or reaction mixture were added to 100 µl of competent *E. coli* cells and left on ice for 30 minutes
2. The cells were heat shocked at 42 °C for 30 seconds, then returned to ice for 30 seconds.
3. 1 ml of SOC media was added.
4. The cells were incubated at 37 °C and 200 rpm for 45 minutes.
5. The tube was spun down at 13000 rpm for 1 minute, the media was removed
6. Bacterial pellet was resuspended in 100 µl of SOC, then plated.

### Lysate preparation

1. Overnight cultures of the transformed bacteria were grown at 37 °C
2. The cultures were transferred to a 10 °C and 200 rpm shaker for 2 hours
3. The cultures were spun down at 4000 rpm for 10 minutes
4. The media was removed, and the pellets were resuspended in

1. For PETases: PBS supplemented with 1mM EDTA and Roche’s cOmplete™ EDTA-free Protease Inhibitor Cocktail, 1 ml of PBS for 10 ml of culture pelleted.
2. For other lysates: 0.4 M Tris-HCl (pH = 7.5), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, EDTA-free protease inhibitor tablet (1 tablet per 50 ml) and 0.1% Triton.

5. The resuspended cells were sonicated at 55% amplitude with 5 seconds of sonication and 10 seconds of rest time for 10 minutes.
6. The tubes were spun down at 4000 rpm at 4 °C for 10 minutes to remove cell debris, the supernatant was kept on ice for immediate use or aliquoted and frozen at -80 °C for storage.

### SDS-PAGE

1. The tank for SDS-PAGE was set up, the inner chamber and half of the outer chamber were filled with 1X running buffer.
2. 8 µl of sample lysate was mixed with 10 µl of 2X Laemmli sample buffer and 2 µl of β-mercaptoethanol under a fume hood.
3. The mixture was boiled at 98 °C for 5 minutes, then spun down and either frozen at -20 °C or loaded onto a precast 4-15% Mini-PROTEAN TGX SDS-PAGE gel together with a protein standard.
4. The power supply unit was connected to the electrodes and run at 180V until desired separation had been achieved.
5. The gel was removed from the tank, carefully broken out of the plastic mould and washed with water.
6. Water was poured off and enough Coomassie Blue to cover the entirety of the gel was added.
7. The gel was incubated while shaking at an orbital shaker at ≈35 rpm until bands were clearly visible.

### Restriction digests

1. For a typical 20 µl reaction using EcoRI, the reaction was set up as follows:

   |  |  |
   | --- | --- |
   | Reagent | Volume or mass added |
   | DNA | 1 µg |
   | 10X reaction buffer | 2 µl |
   | EcoRI | 1 µl |
   | H2O | Up 20 µl |
2. The mixture was incubated at 37 °C for 1 hour.
3. The completed reaction was run on an agarose gel to visualise the products.

### Colony PCR

1. Colonies of interest were picked and resuspended in 50 µl of water.
2. A reaction mixture was set up as below:

   |  |  |
   | --- | --- |
   | Reagent | Volume added |
   | Forward and reverse primers (10 µM) | 0.25 µl |
   | dNTP mix (10 mM) | 0.5 µl |
   | 5X GoTaq reaction buffer | 5 µl |
   | GoTaq polymerase | 0.125 µl |
   | H2O | Up to 25 µl |

   Forward primer: AGGGCGGCGGATTTGTCC; Reverse primer: GCGGCAACCGAGCGTTC
3. The reaction mixture was added to a tube with 10 µl of the cell suspension.
4. PCR was performed as follows:

   |  |  |  |
   | --- | --- | --- |
   | Temperature (°C) | Length | Number of cycles |
   | 95 | 0:03:00 | 1 |
   | 95 | 0:00:15 | 30 |
   | 55 | 0:00:15 |
   | 72 | 0:00:30 |
   | 72 | 0:05:00 | 1 |
5. The completed mixture was run on an agarose gel to visualise whether the desired insert is present.

### Error-prone PCR

1. Dilute primers down to 10 uM
2. Dilute DNA down to 1 ng/uL
3. Set up the reaction using the table below

   |  |  |  |  |  |  |  |  |  |  |
   | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
   | PCR standard buffer | dNTP mix | 55 mM MgCl2 | 0.01 mM MnCl2 | FWD Primer | REV Primer | Taq Polymerase | Plasmid DNA | H2O | Total |
   | 5 uL | 5 uL | 5 uL | 0.5 uL | 1.5 uL | 1.5 uL | 0.5 uL | ~ 5 ng | to 50 uL | 50 uL |

   dNTP concentrations were varied for each base (3.5 mM dATP, 4 mM dCTP, 6mM dGTP, 13.5 mM dTTP) to promote mutations- Then run PCR using the cycling information as follows:

     1 cycle at 95°C for 60s,

     25 cycles at 95°C for 30s,

     1 cycle at 72°C for 45s,

     1 cycle at 68°C for 90s,

     elongation at 68°C for 5 mins then held at 4°C.
   - Mutant MT plasmids were then purified and kept at -18°C.

### Para-Nitrophenol butyrate (pNPB) Assay

1. Prepare 50mM pNPB stock solution and reaction buffer (45 mM Na2HPO4-HCl (pH 7.0), 90 mM NaCl, and 10% (v/v) DMSO)
2. The final reaction system contained protein samples (100-300 µg/ml), 2mM pNPB, and the reaction buffer.
3. The reaction system was incubated for 30 minutes at 250 rpm 37°C for all PETase related constructs

# JUMP Assembly

### Level 0 Assembly

1. The 20 µl reaction mixture was made up as follows:

   |  |  |
   | --- | --- |
   | Reagent | Final concentration |
   | P/R/N/O/C/T DNA part | 1 nM |
   | pJUMP-19 plasmid backbone | 1 nM |
   | 10X T4 ligase buffer | 1X |
   | T4 ligase | 1 U/20 µl |
   | BsaI | 10 U/20 µl |
   | H2O | Up to 20 µl |
2. The mixture was incubated in a thermocycler as outlined below:

   |  |  |  |
   | --- | --- | --- |
   | Temperature (°C) | Length | Number of cycles |
   | 37 | 0:15:00 | 1 |
   | 37 | 0:03:00 | 30 |
   | 16 | 0:03:00 |
   | 55 | 0:15:00 | 1 |
   | 80 | 0:05:00 | 1 |
3. The completed reaction mixture was subsequently used to transform the cells.

### Level 1 Assembly

1. The 20 µl reaction mixture was made up as follows:

   |  |  |
   | --- | --- |
   | Reagent | Final concentration |
   | P/R/N/O/C/T DNA part | 1 nM |
   | pJUMP-19 plasmid backbone | 1 nM |
   | 10X T4 ligase buffer | 1X |
   | T4 ligase | 1 U/20 µl |
   | BsmBI | 10 U/20 µl |
   | H2O | Up to 20 µl |
2. The mixture was incubated in a thermocycler as outlined below:

   |  |  |  |
   | --- | --- | --- |
   | Temperature (°C) | Length | Number of cycles |
   | 42 | 0:15:00 | 1 |
   | 42 | 0:03:00 | 30 |
   | 16 | 0:03:00 |
   | 55 | 0:15:00 | 1 |
   | 80 | 0:05:00 | 1 |
3. The completed reaction mixture was subsequently used to transform the cells.

# *In vitro* Transcription

### Biosensor PCR

The original biosensor DNA was ordered from Twist Biosciences and resuspended in TE buffer. A PCR reaction was setup as follows:

|  |  |
| --- | --- |
| Reagent | Final concentration |
| DNA | 50 pg/µl |
| dNTP mix | 2.5 mM |
| 5X Q5 reaction buffer | 1X |
| Forward and reverse primers | 1.25 mM |
| Q5 DNA polymerase | 1.75 U / 20 µl |
| DMSO | 0.25% |
| H2O | Up to 20 µl |

The PCR reaction was then run as shown in the table below:

|  |  |  |
| --- | --- | --- |
| Temperature (°C) | Length | Number of cycles |
| 95 | 0:05:00 | 1 |
| 95 | 0:00:30 | 30 |
| 67 | 0:00:30 |
| 72 | 0:00:30 |
| 72 | 0:05:00 | 1 |

### DNA transcription

The transcription of biosensors and the binding of the RNA aptamer to the fluorophore were tested using the reaction outlined below:

|  |  |
| --- | --- |
| Reagent | Final concentration |
| DNA | 25 ng/µl |
| DFHBI | 0.1 µM |
| 10X T7 reaction buffer | 1X |
| dNTPs | 1 mM |
| T7 RNA polymerase | 40 U / 20 µl |
| H2O | Up to 20 µl |

The reaction was allowed to proceed in the dark for 1 hour, the tube was then viewed under a blue lightbox to observe fluorescence and loaded onto an agarose gel to visualise any RNA production.

# Protein Immobilization

### Silica Immobilisation

1. The lysates were diluted with 8 pH Tris- HCl to 500 µM.
2. 20 mg of Celite were added to a tube and resuspended in 1 ml of the diluted lysate.
3. The mixture was incubated at 4 °C and 200 rpm for 30 minutes.
4. The tube was centrifuged at 10000 rpm for 10 minutes, the supernatant was discarded.
5. The beads were resuspended in 8 pH Tris-HCl, the mixture was centrifuged at 10000 rpm for 10 minutes and the supernatant was removed.
6. Step 5 was repeated twice.
7. The beads were resuspended in 1 ml PETase buffer (45 mM Na2HPO4, 90 NaCl and 10% (v/v) DMSO).

### Silica-Binding Assay for Characterising Silica Affinity Tags

1. Dilute lysates to the protein concentration of 1 mg/ml using 0.4 M Tris-HCl (pH = 7.5)
2. Add 20 mg of Celite545 into an eppendorf tube and resuspend it in 1 ml of the lysate
3. Incubate the mixtures in a rotary mixer at 4°C for 1 hour.
4. Centrifuge at 10000 rpm for 10 min.
5. Move the supernatants to new tubes.
6. Wash with Tris-HCl (pH=7.5).
7. Assay the fluorescence of the initial (diluted) lysates and final supernatants, using the negative (Tris-HCl) control as the blank.

### Hydrogel production

1. 3% carboxymethyl cellulose solution was prepared by dissolving 30 g of CMC in 750 ml of DI water, stirring at room temperature until solution is even and adding 250 ml of DI water.
2. 20 ml of the stock solution were transferred into a new container and 3 g of citric acid were added while stirring to make a 15% w/v citric acid/CMC solution.
3. The solution was poured into casts and incubated at 40 °C for 24 hours to remove water.
4. After dehydration, the temperature was increased to 80 °C and the mixture was incubated for a further 24 hours to facilitate crosslinking.
5. The hydrogel was dehydrated at 40°C for 24 hours for storage.

### CBD Fusion Protein CMC-Binding Assay

1. Measure initial protein concentration based on Bradford assay.
2. Dilute the lysates to 1 mg/ml by adding Tris-HCl (pH = 7.5)
3. Cut out 20-30 mg of the hydrogel and transfer it to a Falcon tube
4. Add 1 ml of lysate into the tube.
5. Incubate the mixtures in a shaker at 4°C for 1 hour.
6. Transfer the supernatants into new tubes.
7. Assay the fluorescence of the samples, using Tris-HCl (pH = 7.5) as the blank.

### Solubility test

1. From an induced cell culture take 20 ml;
2. Centrifuge cells at 4000 rpm for 5 min;
3. Discard supernatant and freeze pellet at -20ºC for at least 30 min; (if pellet already frozen start from here!)
4. Ressuspend in 2 ml of 1x PBS;
5. Take a 100 µl sample at this point (sample 1);
6. Sonicate in ice (5s pulses and 10s rest pulses);
7. Centrifuge at 4000 rpm for 5 minutes;
8. Take supernatant to a new tube; (sample 2)
9. Resuspend pellet in 2 ml of 1x PBS; (sample 3)
10. To 100 µl of each sample add 100 µl of 2x sample buffer and 10 µl of 2-mercaptoethanol;
11. Run 20 µl in a 10% SDS PAGE gel.

### ICP-MS Sample preparation

1. The CBD-MT and lacZ lysates were thawed out on ice and diluted with 7.5 pH Tris-HCl to a protein concentration of 1 mg/ml.
2. 1 ml of a diluted lysate was added to each hydrogel as shown in the following table.

   | Hydrogel number | Lysate added | Metal added |
   | --- | --- | --- |
   | 1 | lacZ | None |
   | 2 | lacZ | None |
   | 3 | lacZ | Zn2+ |
   | 4 | lacZ | Zn2+ |
   | 5 | lacZ | Zn2+ |
   | 6 | lacZ | Ni2+ |
   | 7 | lacZ | Ni2+ |
   | 8 | lacZ | Ni2+ |
   | 9 | CBD-MT | None |
   | 10 | CBD-MT | None |
   | 11 | CBD-MT | Zn2+ |
   | 12 | CBD-MT | Zn2+ |
   | 13 | CBD-MT | Zn2+ |
   | 14 | CBD-MT | Ni2+ |
   | 15 | CBD-MT | Ni2+ |
   | 16 | CBD-MT | Ni2+ |
3. The hydrogels were incubated at a shaker for 1 hour at 4 °C and 150 rpm.
4. The supernatant was discarded, and the hydrogels were washed with 7.5 pH Tris-HCl thrice.
5. 1 ml of 7.5 pH Tris-HCl with either 100 µM Ni2+, 100 µM Zn2+, or containing no metal, was added to the hydrogel as shown in table 1.
6. The hydrogels were incubated at a shaker for 1 hour at 37 °C and 200 rpm.
7. 1 ml of supernatant from each tube was transferred into an Eppendorf tube for analysis using ICP-MS.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Contents

* [1) Biosensing](#1)
  + [1.1) Linear Biosensors](#1.1)
  + [1.2) Transcription Factors](#1.2)
  + [1.3) *In vitro* transcription](#1.3)
* [2) Bioremediation](#2)
  + [2.1) Initial characterization](#2.1)
  + [2.2) Protein production](#2.2)
  + [2.3) Characterizing protein-hydrogel interaction](#2.3)
  + [2.4) Metallothionein displaying 3C-Hydrogels](#2.4)
  + [2.5) Directed Evolution](#2.5)
* [3) Bioconversion](#3)
  + [3.1) Assembly](#3.1)
  + [3.2) Protein expression and solubility test](#3.2)
  + [3.3) Immobilisation and Activity assessment](#3.3)

[![](https://static.igem.wiki/teams/4390/wiki/138543-200.png)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

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# **Biosensing**

---

### 1.1) Linear Biosensors

##### PCR Amplification of linear biosensors from G-blocks

| Heavy metal transcription factor binding site | RNA aptamer | Link to parts repository |
| --- | --- | --- |
| MerR | Broccoli | [BBa\_K4390099](http://parts.igem.org/Part%3ABBa_K4390099) |
| MerR | iSpinach | [BBa\_K4390097](http://parts.igem.org/Part%3ABBa_K4390097) |
| MerR | Squash | [BBa\_K4390098](http://parts.igem.org/Part%3ABBa_K4390098) |
| ArsR | Broccoli | [BBa\_K4390105](http://parts.igem.org/Part%3ABBa_K4390105) |
| ArsR | iSpinach | [BBa\_K4390100](http://parts.igem.org/Part%3ABBa_K4390100) |
| ArsR | Squash | [BBa\_K4390104](http://parts.igem.org/Part%3ABBa_K4390104) |
| PbrR | Broccoli | [BBa\_K4390102](http://parts.igem.org/Part%3ABBa_K4390102) |
| PbrR | iSpinach | [BBa\_K4390106](http://parts.igem.org/Part%3ABBa_K4390106) |
| PbrR | Squash | [BBa\_K4390101](http://parts.igem.org/Part%3ABBa_K4390101) |

**Table 1.** Shows all nine linear biosensors designs with their heavy metal transcription factor (MerR = cadmium and mercury biosensor ArsR = arsenic biosensor PbrR = lead biosensor) and what RNA aptamer they transcribed. Also, contains links to the parts repository for each biosensor so you can see the exact sequence that was ordered from IDT. For more information go to our [Design page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D).

![](https://static.igem.wiki/teams/4390/wiki/results/figure-1-biosesnors.png)

**Figure 1.** Agarose gel electrophoresis of PCR amplification product of all our linear biosensors. F30 flanked RNA aptamers were used as the controls (Broccoli:  [Part:BBa\_K4390095](parts.igem.org/Part%3ABBa_K4390095) Squash: [Part:BBa\_K4390096](parts.igem.org/Part%3ABBa_K4390096) iSpinach: [Part:BBa\_K4390094](parts.igem.org/Part%3ABBa_K4390096)). 3µl of PCR reaction mixture was loaded into each well. (Br = Broccoli, iS = iSpinach SQ = Squash CON = constitutive)

All our linear biosensors (Table 1) had been ordered in whole as G-blocks from IDT. To ensure we had enough DNA for the later in-vitro transcription experiments, we performed a PCR amplification (Figure 1). The linear DNA was purified from the PCR reactions. After the purification, the DNA concentration in the tubes for all the linear construct was around 30-50 ng/µl, indicating that the PCR worked for all the constructs. However, when we ran the gel, we did not see any band for the mercury iSpinach construct (Hg iS). This show that the DNA could not be amplified, and the result is most likely an anomaly. Bands for all the other biosensors were present, however, the arsenic Broccoli biosensor (Ars Br), the arsenic iSpinach construct (Ars iS) and the lead Broccoli (Pb Br) all had very faint bands suggesting a low yield of DNA. We hypothesise that the low yield of DNA from PCR was because of secondary structures spontaneously formed by the DNA, as adding DMSO to the reaction mixture increased PCR efficiency. The extracted DNA was used for running a biosensor test.

### 1.2) Transcription Factors

For more information on the design of our transcription factor plasmids go to our [Design page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D).

##### Construction of level 1 plasmids

![](https://static.igem.wiki/teams/4390/wiki/results/figure-2-biosesnors.png)

**Figure 2.**Agarose gel electrophoresis for EcoR1 digest for the mutated MerR (mMerR), PbrR and ArsR transcription factor level 1 JUMP plasmids.

![](https://static.igem.wiki/teams/4390/wiki/results/figure-3-biosensors.png)

**Figure 3.** Agarose gel electrophoresis of colony PCR a) level 1 MerR JUMP assembly b) level 1 ArsR and mutated MerR (m\_MerR) JUMP assemblies.

After we had completed our level one assemblies for the mutated MerR (m\_MerR), PbrR and ArsR transcription factor level one plasmids we ran a digestion on the plasmids using EcoR1 (Figure 2). The mutated MerR and the ArsR plasmids were around 3000bp, suggesting that the acceptor plasmid was ligated without the transcription factor insert. Only the PbrR was assembled correctly as we see a band around 3500bp which is the size band we expect if there is an insert in the plasmid. We then performed the m\_MerR and ArsR assemblies again along with the MerR. We were unable to do this assembly along with the initial ones we did as we did not have the parts needed to make this assembly. We then ran a colony PCR on these three assemblies to check for the presence of our insert in the plasmids. In figure 3a we can see a smear at around 400bp which suggests that the level 1 JUMP plasmid contains the MerR insert. However, for the ArsR and mutated MerR, we saw no band (Figure 3b) showing that there was no insert present in the DNA and that the assembly failed. Due to time constraints, we could no longer pursue the ArsR and mutated MerR and try and get a working assembly. So, we only used the MerR and PbrR assemblies for protein expression analysis.

##### Protein Expression Analysis

To perform the protein expression analysis we transformed *E. coli* Bl21(DE3) with the level 1 JUMP containing the PbrR and MerR expression cassettes and performed SDS-PAGE analysis (Figure 4). The SDS-PAGE indicated that both expression cassettes produced their respective transcription factors. The presence of the band at 15 kDa in the PbrR lane indicates that it expresses PbrR as this is its predicted size. We also saw a band at 16 kDa in the MerR lane which indicates that it expresses MerR as this is its predicted size.

![](https://static.igem.wiki/teams/4390/wiki/diagrams/image-20221013-163208-e46ddb24.png)

**Figure 4.** SDS-PAGE of the lysates of *E. coli* BL21(DE3) which were transformed with the level 1 JUMP plasmids containing the PbrR and MerR expression cassettes. Loading was standardised using a Bradford assay, with 3 mg of proteins loaded into each well. Arrows show the band which indicates we have expression of our transcription factors.

### 1.3) *In vitro* transcription

Once we had our linear construct and cells which expressed the transcription factors we then wanted to characterise our biosensors. To do this we performed our in-vitro transcription reactions (More information on the experiments page). We did not include the cell lysates containing the transcription factors as we wanted to see if the linear constructs would produce fluorescence without the transcriptional repression caused by the transcription factors (More information on the design page). However, none of our linear biosensors produced any fluorescence (Figure 5a) when compared to our positive control of GFP seen at the top of figure 5a. This would suggest either that there is no RNA transcription happening or that the RNA aptamer is unable to bind to the DFHBI fluorophore as it is no longer functional.

In normal light conditions after 2 hours, we noticed a slight colour change from clear to green in all our experimental in-vitro transcription reactions (Figure 5b 5c). This colour change cannot be observed under blue light and is therefore most likely a by-product of the DFHBI fluorophore. This would suggest that the fluorophore is still functional and that no RNA transcription is taking place.

To confirm this we performed a gel electrophoresis on the in-vitro transcription factor reactions (Figure 6) to see if we could detect any RNA being produced in the reactions. In the gel, we can clearly see there is DNA present, however, we could not see any RNA smear below the DNA, confirming that no RNA transcription is taking place.

![](https://static.igem.wiki/teams/4390/wiki/results/figure-5-biosense.png)

**Figure 5.** Images of the in-vitro transcription reactions after 2 hours a) observed under a Safe Imager™ Blue-Light Transilluminator (Invitrogen) with an amber filter unit and imaged using a phone camera along with a postive control of GFP b) observed under normal light and imaged using a phone camera with a postive control of GFP (Top left) and negative control containing no template DNA (Top right) c) close up of in-vitro transcription reaction using the lead iSpinach construct (Right) and negative control with no template DNA (left).

![](https://static.igem.wiki/teams/4390/wiki/results/screenshot-2022-10-11-at-21-08-36.png)

**Figure 6.** Agarose gel electrophoresis of in-vitro transcription reactions (Br = Broccoli, iS = iSpinach SQ = Squash CON = Constitutive).

To troubleshoot this problem we tried replacing all our reagents with reagents from other labs to see if they were preventing RNA transcription. However, we still could not produce fluorescence. The only reagent we could not replace from another lab was the DFHBI fluorophore. However, as we had no RNA transcription this would likely not be the cause of the problem. If we did not have time and cost restraints we would of ordered in fresh reagents to ensure that these were no the problem.

We also tried using a linear biosensor construct which is known to work. The construct was kindly given to us by Mengxi Li who works on cell-free biosensors similar to ours. However, even using her construct we were still unable to produce fluorescence. Mengxi also looked at our protocols and construct design and could see nothing wrong with them. This meant that we were unable to find the exact cause of our problem.

# **Bioremediation**

---

### 2.1) Initial characterization

After synthesis and dehydration of our CMC-CA hydrogels, which are composed of sodium carboxymethylcellulose (CMC) and the citric acid (CA) crosslinker, we tested their properties and viability in water by doing swelling and degradation tests. For verifying the presence of citric acid-mediated cross-linking, we compared them to negative control ‘hydrogels’ made only with CMC. We did these tests on two different sizes and shapes of the CMC-CA hydrogel: 25 ml frog-shaped hydrogels and 1 ml box-shaped hydrogels (Figure 7).

![](https://static.igem.wiki/teams/4390/wiki/results/figure-1-biorem.png)

**Figure 7.** Initial characteristics of the hydrogels after synthesis and dehydration. (A) The 1st and 3rd rows show the negative control ‘hydrogels’ made of CMC only and the 2nd and 4th rows show the CMC-CA hydrogels made from 3% w/v sodium carboxymethylcellulose (CMC) and 15% w/v citric acid (CA), (B) Initial weights (W0) of the hydrogels before the swelling and degradation tests. The CMC-CA hydrogels had significantly larger weights than the negative control ‘hydrogels’ despite being made from the same volumes in the same moulds.

When making the hydrogels we noticed that the control samples were thin and very fragile (easy to break when being handled) compared to the slightly thicker and more rigid CMC-CA hydrogels. The 25 ml frog-shaped hydrogels were quite fragile and fragmented while being removed from the silicon moulds probably due to the irregular mould shape and the large number of bubbles that emerged during the dehydration process.

##### Swelling Tests

After immersion in water for 1 hour, the swollen weights (WS) of the hydrogels were measured and compared to the initial weight of the hydrogels (W0) (Figure 8).

![](https://static.igem.wiki/teams/4390/wiki/results/figure-2a-biorem.png)

![](https://static.igem.wiki/teams/4390/wiki/results/figure-2b-biorem.png)

**Figure 8.** Swelling test results for our CMC-CA hydrogels. (A) Images showing the CMC and CMC-CA hydrogels immersed in water after T=0 minutes, T = 30 minutes and T = 60 minutes (B) Shows the swelling degree of the CMC-CA hydrogels of the two different shaped hydrogels. The swelling degree was calculated using the following equation:  Swelling Degree (SD%) = ((WS-W0)/W0) x 100%

After 30 minutes of being underwater, the negative control ‘hydrogels’ had already completely disintegrated and dissolved (Figure 2A), so no measurements could be taken for them. This is because CMC is soluble in water and there was no citric acid added to the negative controls to prevent this. Therefore, this test also confirmed the presence of citric acid-mediated crosslinking in the CMC-CA hydrogels. This proves that the citric acid crosslinking is what makes the hydrogels insoluble.

The 25ml frog-shaped CMC-CA hydrogels were able to take on water and showed a 32% increase in weight whilst the 1ml box-shaped CMC-CA hydrogels did not take on any water and had a 2% loss of weight. We think that either something went wrong in their construction or that their small size prevented them from taking on water.

##### Degradation tests

After the swelling tests, the hydrogels were re-dehydrated at 30-40°C overnight, then the final weights (WF) of the hydrogels were measured and compared to the initial weight of the hydrogels (W0) (Figure 9).

![](https://static.igem.wiki/teams/4390/wiki/results/fig-3a-biorem.png)

![](https://static.igem.wiki/teams/4390/wiki/results/screenshot-2022-10-11-at-21-46-32.png)

**Figure 9.** Degradation test results for our CMC-CA hydrogels. (A) Images showing the CMC-CA hydrogels after being re-dehydrated overnight. 25ml frog shaped hydrogel top, 1ml box-shaped bottom (B) Shows the degradation degree of the CMC-CA hydrogels of the two different shaped hydrogels. The degradation degree was calculated using the following equation:  Degradation Degree (DD%) = ((W0-WF)/W0) x 100%

Both shaped showed significant damage (Figure 9A) and weight loss after re-dehydration as they both showed degradation degrees of around 77% (Figure 9B). This shows that the CMC-CA hydrogels could not be reused after being immersed in water and if they were to be used for a bioremediation device they could only be used once and then recycled. However, this result was expected as the hydrogels swell when submerged in water as the hydrophilic carboxyl groups interact with the water molecules, creating enough tension to maintain the water inside the polymeric mesh. However, as not all the carboxymethylcellulose or citric acid is crosslinked and incorporated into the network of the hydrogel proper. Therefore, when submerged in water the reagents that hadn’t reacted are washed out of the hydrogel. This causes a loss of mass or degradation.

### 2.2) Protein production

##### Production of CBD- and SB7-tagged Proteins and Non-Tagged sfGFP

After we successfully produced our level 1 JUMP assemblies for our CBD- and SB7-tagged proteins (SB7-sfGFP, CBD-Metallothionein (MT) and CBD-sfGFP) We also produced non-tagged sfGFP to use as controls for the fluorescence assays for assessing the performance of the CBD and SB7 tags. We then transformed them into *E. coli* BL21(DE3) for protein expression. After protein expression in the BL21(DE3) cell cultures, the cultures were lysed by sonication, and the lysates were run on an SDS-PAGE gel to confirm the presence of our CBD- and SB7-tagged proteins (Figure 10). This SDS-PAGE step also serves as a solubility test to confirm that all of our desired proteins are in the soluble fraction and can be properly incorporated into protein immobilisation methods.

![](https://static.igem.wiki/teams/4390/wiki/results/figure-4-biorem.png)

**Figure 10.** SDS-PAGE gel of the lysates containing our expressed constructs. Lane 0 represents the negative control, which is the BL21(DE3) strain containing only the pJUMP29 LacZ acceptor plasmid without any insert. The red lines indicate the bands representing our constructs. The ladder we used was Prestained Protein Marker, Broad Range (7-175 kDa) (NEB #P7708S).

As we saw bands at all the correct sizes on our SDS-PAGE (indicated by the red lines in Figure 10) it proved that all our cells expressed our level 1 JUMP assemblies.

### 2.3) Characterizing protein-hydrogel interaction

##### Characterising CBD Interaction with CMC-CA Hydrogels (and Comparison to Silica Immobilisation Method)

We tested the interaction of our CBD tag with our CMC-CA hydrogels by fluorescence assays of our lysates containing sfGFP constructs. We expected the fluorescence intensity of the CBD-sfGFP lysate to decrease after incubating the hydrogels in them as the protein tag could interfere with the sfGFP. We also compared the effectiveness of CBD-sfGFP binding to our hydrogels compared to SB7-sfGFP binding to Celite545 silica beads (20-100 um in diameter). Before the fluorescence assays were carried out, we measured the total protein concentration of our lysates, and then diluted all of them to 1.0 mg/ml total protein concentration for uniform measurement.

| Lysate | Initial Fluorescence Intensity |
| --- | --- |
| Non-tagged sfGFP | 40813 |
| CBD-sfGFP | 19223 |
| SB7-sfGFP | 13268 |

**Table 2.** Initial fluorescence intensities of lysates containing sfGFP constructs. All lysates were diluted to 1.0 mg/ml total protein concentration using the buffer (0.4 M Tris-HCl, pH 7.5) to allow for uniform measurement environments.

Table 2 shows that the non-tagged sfGFP lysate has a significantly higher initial fluorescence intensity than the CBD-sfGFP and SB7-sfGFP, suggesting that N-terminal CBD- and SB7-tagging of the sfGFP may affect its folding and thus, have negative impacts on protein function.

##### CBD Binding to CMC-CA Hydrogel Matrix

To determine level of CBD-sfGFP interaction with our cellulose-based solid materials (or SB7-sfGFP interaction with silica beads), we measured the proportion of the sfGFP constructs bound to the solids (or pellets) compared to the remaining cell lysate.

We took 20-30 mg fragments from the 1 ml box-shaped hydrogels (it was very difficult to cut out fragments of equal weight because we lacked precision cutting instruments). Due to the varying weights of our hydrogel fragments, we divided the data obtained for each of them by its weight and multiplied this by 20 to calculate the level of CBD binding to 20 mg of the hydrogel as follows:

%sfGFP Construct Bound = ((I0-IF) x 20/hydrogel weight)/I0) x 100%

Where I0 denotes the initial fluorescence intensity of the lysate and IF denotes the final fluorescence intensity of the supernatant after incubation of the cellulose/silica-based solids for 1 hour.

These fragments were incubated in 1 ml of lysate containing non-tagged sfGFP (control) or CBD-sfGFP for 1 hour, then they were removed. Figure 5 shows the fluorescence assay results.

![](https://static.igem.wiki/teams/4390/wiki/results/figure-5-biorem.png)

**Figure 11.** Percentages of non-tagged sfGFP or CBD-sfGFP bound to a 20 mg CMC-CA hydrogel fragment incubated in 1 ml of their respective lysates (1.0 mg/ml total protein concentration).

Oddly, there was no significant difference in the levels of immobilisation between the non-tagged sfGFP control and our CBD-sfGFP construct, but it is worth noting that the hydrogels bound significant amounts of both sfGFP constructs (70-75%). This may suggest that the hydrogels have a significant level of non-selective absorption of proteins.

To confirm this superabsorbent property, we measured the total protein concentrations in the final supernatants and calculated the percentage of protein absorbed into the hydrogels from the lysates. This experiment revealed that a 20 mg CMC-CA hydrogel fragment absorbs, on average, 52.62% total protein content from a 1 ml lysate (with an initial total protein concentration of 1.0 mg/ml). This confirms the high levels of non-specific protein interactions with the hydrogels and so, we proceeded to alter the CBD binding test by substituting the CMC-CA hydrogel matrix with non-absorbent microcrystalline cellulose to better characterise selective CBD affinity to cellulose-based solid platforms.

##### CBD Binding to Microcrystalline Cellulose

20 mg of insoluble microcrystalline cellulose were incubated in 1 ml of lysate (of 1.0 mg/ml total protein concentration) containing non-tagged sfGFP (control) or CBD-sfGFP for 1 hour, then the supernatants were isolated from the pellets by centrifugation (Figure 12).

![](https://static.igem.wiki/teams/4390/wiki/results/screenshot-2022-10-11-at-21-47-09.png)

**Figure 12.** Non-tagged sfGFP or CBD-sfGFP bound to 20 mg Avicel microcrystalline cellulose incubated in 1 ml of their respective lysates (1.0 mg/ml total protein concentration). (A) Shows the percentage difference of constructs bound measured by fluorescence intensity of the microcrystalline cellulose (B) images of our fluorescence results with untagged sfGFP on the left and the CBD tagged sfGFP on the right obtained using a blue light box.

The percentage of CBD-sfGFP immobilised is 81% while that of non-tagged sfGFP control is only 10% (Figure 12A) and we also saw visibly more intense fluorescence in the lysate containing CBD-sfGFP (CBD) than the control (no CBD). This proves that our CBD tag selectively binds to cellulose and immobilises the protein fused to it. This can also be applied for the CMC-CA hydrogel matrix, because the CBD specifically binds to the beta-1,4-glycosydic linkage of the cellulose and CMC sugar backbone.

##### SB7 Binding to Silica Beads

20 mg of Celite545 silica beads were incubated in 1 ml of lysate containing non-tagged sfGFP (control) or SB7-sfGFP for 1 hour, then the supernatants were isolated from the pellets by centrifugation (Table 3).

![](https://static.igem.wiki/teams/4390/wiki/results/table-2-biorem.png)

**Table 3.** Percentages of non-tagged sfGFP or CBD-sfGFP bound to a 20 mg Celite545 silica beads incubated in 1 ml of their respective lysates (1.0 mg/ml total protein concentration).

The values were oddly significantly negative, but we would assume virtually 0% of both sfGFP constructs were immobilised to silica beads. Therefore, our SB7 tag failed to immobilise proteins to silica beads. This may be due to something wrong with the design of the SB7 tag sequence.

### 2.4) Metallothionein displaying 3C-Hydrogels

##### Zinc (II) and Nickel (II) Binding Capacity of Metallothionein-Displaying 3C Hydrogels

Our metallothionein-displaying 3C hydrogels were prepared by incubating 20-30 mg fragments of the (1 ml, box-shaped) CMC-CA hydrogels in 1 ml of the lysate (of 1.0 mg/ml total protein concentration) containing CBD-MT for 1 hour. For the negative controls, CMC-CA hydrogel fragments were incubated in 1 ml of lysate of the negative control BL21(DE3) hosting the pJUMP29 backbone plasmid with no insert.

The initial and final metal ion concentrations in the supernatants were measured by inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS measures the concentration of a certain metal ion in solution. Therefore, the reduction in the number of unbound metal ions in the supernatant represents the number of metal ions sequestered in the 3C hydrogels (Figure 13).

![](https://static.igem.wiki/teams/4390/wiki/results/figure-7-biorem.png)

**Figure 13.** Amounts of (A) Zn (II) and (B) Ni (II) ions captured by 3C hydrogels. The 3C hydrogel fragments were washed with the buffer (0.4 M Tris-HCl, pH 7.5) 3 times, then incubated in 1 ml of 100 uM Zn (II) or Ni (II) solution (using the buffer as the solvent). The values were obtained by multiplying the molecular weight of the metal ion with the difference between the initial and final concentrations of metal ion in the supernatant, which were measured using ICP-MS. Because of varying weights of the hydrogel fragments, the quantity of metal ion sequestered was divided by the weight of the hydrogel fragment used and multiplied by 20 to calculate the data representative of a 20 mg hydrogel fragment.

We saw no significant difference between the control and the 3C hydrogel decorated with CBD-tagged metallothionein. The most probable reason for this would be that the hydrogel matrix plays a major role in metal ion sequestration, as we saw this effect in earlier experiments. Another reason may be that the CBD tag is affecting the metallothionein folding and therefore reducing its metal binding capacity, we also saw this effect in earlier results.

If we were to pursue this further, we would use purified proteins instead of crude lysates as they are too impure and contain metalloenzymes and metal-chelating proteins which could affect our results. We could also try to optimise the expression and purification of CBD-tagged proteins to ensure correct folding and so, allow effective solid-phase immobilisation while maintaining protein function.

### Directed Evolution

##### Determination of the Maximum Inhibitory Concentration

We established the Maximum Inhibitory Concentration (MIC) of untransformed BL21(DE3) and TOP10 cells by plating on agar plates with a range of AgNO3 concentrations, starting with 8 mg/L until 16 mg/L, where we observed a fall-off in growth of BL21(DE3) cells (See figure 14)

![](https://static.igem.wiki/teams/4390/wiki/results/screenshot-2022-10-12-at-00-19-25.png)

**Figure 14.** Growth of Native *E. coli* strains on Ag+ containing LB agar plates. Grey boxes indicate a lawn, white boxes indicate no growth, otherwise colony counts are specified.

With this, we had determined that the MIC of AgNO3 for BL21(DE3) cells was 16mg/L, as there was a growth fall off afterwards.

##### Screening metallothionein mutants

Next we used error-prone PCR to generate mutants of *M. edulis, M. galloprovincialis, D. rerio*, and *S. cerevisiae* MTs. We made transcriptional units with the error prone PCR product, and found the following colony counts (See figure 15).

![](https://static.igem.wiki/teams/4390/wiki/results/screenshot-2022-10-12-at-00-25-53.png)

**Figure 15.** a) Illustrates, colony counts of BL21(DE3) recombinantly expressing MTs. In the figure, BL21 (DE3) cells expressing MTs of *Danio rerio* (DR), *Saccharomyces cerevisiae* (SC), *Mytilus galloprovincialis* (MG), and *Mytilus edulis* (ME) were plated, the wild type is shown in grey and the randomly picked mutant (EP) in red. The figure shows a significant difference in MIC between all the MTs expressing cells and the control. The figure doesn’t indicate if the mutants were better than WTs or vice versa. However, some MTs grew at higher concentration but did not grow at lower concentrations. b) Shows an example of the raw data using WT MT DR plates.

MTs of almost all species except for *S. cerevisiae* and *M. galloprovincialis* grew as lawns at 16 mg/L and 17 mg/L AgNO3. We were not able to do any screening, however we were able to propose a new selection method for MT function. Since no mutants were screened, we did not use the directed evolution mutants in the rest of our bioremediation device.

Our iGEM Team also took part in the iDEC Competition (International Directed Evolution Competition). To read more in depth about our directed evolution exploration, check out our [wiki](https://idec-teams.github.io/2022_Edinburgh-UHAS_Ghana/).

# **PET Bioconversion**

---

### 3.1) Assembly

##### Overview of all planned constructs results

17 of 22 of planned Lv.1 JUMP plasmids were successfully constructed (See Figure 16). The enzyme solubility or activity were assessed. All the successful PETase-related constructs were immobilised, and the activity of them was assessed. We chose the best composite part [C-terminal L2NC-linker-tagged FAST-PETase] for the proof-of-concept.

![](https://static.igem.wiki/teams/4390/wiki/results/fig-1-pet.png)

**Figure 16.** The overview of each composite part result for PET biodegradation. T7pro: T7 promoter; B0034:B0034 ribosome binding site; L1U1H08: Terminator L1U1H08.

##### Lv.1 JUMP plasmid construction

After JUMP assembly, successful assemblies were selected from Blue-White Screening and further verified by colony PCR. The successful assemblies are listed below in Table 4:

| Part Name | Shorthand name | Link to parts repository |
| --- | --- | --- |
| Untagged Dou-PETase | [Dou-PETase] | [BBa\_K4390088](http://parts.igem.org/Part%3ABBa_K4390088) |
| N-terminal Car9-tagged Dou-PETase | Car9-linker-[Dou\_PETase] | [BBa\_K4390085](http://parts.igem.org/Part%3ABBa_K4390085) |
| Untagged Tri-PETase | [Tri\_PETase] | [BBa\_K4390089](http://parts.igem.org/Part%3ABBa_K4390089) |
| N-terminal L2NC-linker-tagged Tri-PETase | L2NC-linker-[Tri\_PETase] | [BBa\_K4390116](http://parts.igem.org/Part%3ABBa_K4390116) |
| N-terminal Car9-tagged Tri-PETase | Car9-linker-[Tri\_PETase] | [BBa\_K4390086](http://parts.igem.org/Part%3ABBa_K4390086) |
| C-terminal L2NC-tagged Tri-PETase | [Tri\_PETase]-L2NC | [BBa\_K4390082](http://parts.igem.org/Part%3ABBa_K4390082) |
| C-terminal L2NC-linker-tagged Tri-PETase | [Tri\_PETase]-linker-L2NC | [BBa\_K4390083](http://parts.igem.org/Part%3ABBa_K4390083) |
| C-terminal Car9-tagged Tri-PETase | [Tri\_PETase]-linker-Car9 | [BBa\_K4390081](http://parts.igem.org/Part%3ABBa_K4390081) |
| Untagged FAST-PETase | [FAST\_PETase] | [BBa\_K4390090](http://parts.igem.org/Part%3ABBa_K4390090) |
| N-terminal L2NC-linker-tagged FAST-PETase | L2NC-linker-[FAST\_PETase] | [BBa\_K4390114](http://parts.igem.org/Part%3ABBa_K4390114) |
| N-terminal Car9-tagged FAST-PETase | Car9-linker-[FAST\_PETase] | [BBa\_K4390084](http://parts.igem.org/Part%3ABBa_K4390084) |
| C-terminal L2NC-tagged FAST-PETase | [FAST\_PETase]-L2NC | [BBa\_K4390076](http://parts.igem.org/Part%3ABBa_K4390076) |
| C-terminal L2NC-linker-tagged FAST-PETase | [FAST\_PETase]-linker-L2NC | [BBa\_K4390077](http://parts.igem.org/Part%3ABBa_K4390077) |
| C-terminal Car9-tagged FAST-PETase | [FAST\_PETase]-linker-Car9 | [BBa\_K4390075](http://parts.igem.org/Part%3ABBa_K4390075) |
| Untagged MHETase | [MHETase] | [BBa\_K4390091](http://parts.igem.org/Part%3ABBa_K4390091) |
| N-terminal L2NC-linker-tagged MHETase | L2NC-linker-[MHETase] | [BBa\_K4390118](http://parts.igem.org/Part%3ABBa_K4390118) |
| N-terminal Car9-tagged MHETase | Car9-linker-[MHETase] | [BBa\_K4390087](http://parts.igem.org/Part%3ABBa_K4390087) |

**Table 4.** Successful JUMP assemblies related to PETase immobilisation on silica beads.

The 4 assemblies with N-terminal L2NC silica tag failed (Figures 17 and 18). We tried to move the L2NC DNA fragment from plasmid containing C-terminal-L2NC sequence to new plasmids for constructing N-terminal-L2NC silica tag. However, the PCR of the C-terminal-L2NC silica tag failed.

![](https://static.igem.wiki/teams/4390/wiki/results/fig-2-pet.png)

**Figure 17.** Agarose gel shows the PCR result of L2NC silica tag (agarose concentration 1.2%). The left lane was loaded with L2NC PCR product. The ladder used on the right lane was: 1 kb DNA Ladder from NEB (N3232S)

![](https://static.igem.wiki/teams/4390/wiki/results/fig-3-pet.png)

**Figure 18.** Agarose gel shows the PCR result of [L2NC-linker-Dou\_PETase] (agarose concentration 1.2%). The B1 and B2 lanes were loaded with PCR product. The lanes were labelled with letters, and the number behind each letter represented different colonies from Blue-White Screening. B: [T7Pro-B0034-L2NC-linker-[Dou-PETase]-L1U1H08]. The L Lane was load with 1 kb DNA Ladder from NEB (N3232S).

### 3.2) Protein expression and solubility test

The Lv.1 plasmids were transformed into *E. coli* SHuffle strains for protein expression. We then ran an SDS-PAGE to see if our proteins were subsequently expressed (Figure 19). This SDS-PAGE step also serves as a solubility test to confirm that all our desired proteins are in the soluble fraction and can be properly incorporated into protein immobilisation methods (Figure 20).

![](https://static.igem.wiki/teams/4390/wiki/diagrams/figure-19.jpg)

**Figure 19.** The solubility test result of different constructs. The soluble portions of each construct cell lysates after centrifuge were load on the gel. The lanes were labelled with letters representing different constructs. C: Car9-linker-[Dou\_PETase]. D: [Tri\_PETase]. E: L2NC-linker-[Tri\_PETase]. F: Car9-linker-[Tri\_PETase]. G: [Tri\_PETase]-L2NC. H: [Tri\_PETase]-linker-L2NC. I: [Tri\_PETase]-linker-Car9. The ladder was Prestained Protein Marker, Broad Range (7-175 kDa) (NEB #P7708S), and the range of constructs weight was labelled.

![](https://static.igem.wiki/teams/4390/wiki/diagrams/figure-20.png)

**Figure 20.** The solubility test result of different constructs. The soluble portions of each construct cell lysates after centrifuge were load on the gel. The lanes were labelled with letters representing different constructs. S: [MHETase]. T: L2NC-linker-[MHETase]. V: Car9-linker-[MHETase]. The ladder used: P7718S protein ladder from NEB, and the range of constructs weight was labelled. - control was the cell lysates of protein expressing strain without any Lv.1 plasmids.

From Figures 19 and 20, bands with correct weights were observed (labelled with a red line). This confirmed that we had successfully expressed untagged PETase, Car9-PETase, L2NC-linker-PETase, untagged MHETase, Car9-MHETase and L2NC-linker-MHETase.

### 3.3) Immobilisation and Activity assessment

##### PETase-silica tag fusion protein Activity Test

We assessed the PETase mutants' activity based on para-nitrophenol-butyrate (pNPB) assay, since pNPB can be hydrolysed by PETase into para-nitrophenol (pNP) with maximum absorbance at 415 nm. This is a preliminary assay to determine the activity of PETase, although pNPB has structural differences to the polyethylene terephthalate which is the real substrate of PETase.

First, we assessed the FAST-PETase constructs activity towards pNPB, comparing to Dou-PETase (Figure 21). Dou-PETase is the old part from iGEM repository ([BBa\_K3946023](parts.igem.org/Part%3ABBa_K3946023)).

![](https://static.igem.wiki/teams/4390/wiki/results/figure-6-pet.png)

**Figure 21.** The protein sample activities assessment based on para-nitrophenol-butyrate pNPB assay. The reaction system was set up in 96 wells plate with final volume 10ul in each well, and the reaction continued for 30 min in 37°C (45 mM Na2HPO4-HCl (pH 7.0) 90 mM NaCl, and 10% (v/v) DMSO; 2mM pNPB-para-nitrophenol butyrate). A415 was measured on the plate reader (FLUOstar Omega). All the constructs were constructed with T7 promoter, B0034 RBS and L1U1H08 terminator, so only the shorten forms of construct name were on the X-axis. [Shuffle without Lv.1 plasmid. 1] is the protein sample from the empty Shuffle strain of the same batch. “U” is the amount of activity which releases one micromole of pNP per minute under these assay conditions. The standard deviation is derived from biological triplicates.

From the first set of pNPB assay (Figure 21), we observed that the protein sample containing untagged FAST-PETase showed the highest activity towards pNPB under the reaction condition over protein samples containing other constructs and empty control. All protein samples containing FAST-PETase-silica tag fusion proteins, except [L2NC-linker-FAST\_PETase], showed activity towards pNPB higher than the empty control. The FAST-PETase-silica-tag fusion proteins showed similar activities as the untagged Dou-PETase. The empty control still had measurable activity towards pNPB, which supposed the *E. coli* SHuffle strain constitutively expresses certain Esterases that can hydrolyse pNPB. Generally, C-terminal tagged FAST-PETase showed higher activity than the N-terminal tagged ones. We supposed this may be due to elongated N-terminal flexible structure may interact and inhibit the FAST-PETase activity towards pNPB.

We found the 96 wells plate and plate reader were not suitable to assess the PETase activity after they immobilized on the actual silica beads. Therefore, we changed the reaction device to 1.5ml Eppendorf tube and spectrometer. Data for Tri-PETase and FAST-PETase were measured across different days (Figure 22). We observed an inconsistency in the empty control activity towards pNPB across different days. Therefore, we calculated the fold-change of individual protein sample activity towards the empty control in the same batch to reduce the inconsistency when comparing the data.

![](https://static.igem.wiki/teams/4390/wiki/diagrams/new-figure-22-on-results.png)

**Figure 22.** The protein sample activities result based on para-nitrophenol-butyrate pNPB assay. The figure presented the fold-change of protein samples activity over the activity in empty control from the same batch. The fold changes of activity from [Tri\_PETase] to [Tri\_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.2]. The fold changes of activity from [FAST\_PETase] to [FAST\_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.3]. The reaction system was set up with final volume 1ml in each Eppendorf tube, and the reaction continued for 30 min in 37°C (45 mM Na2HPO4-HCl (pH 7.0) 90 mM NaCl, and 10% (v/v) DMSO; 2mM pNPB-para-nitrophenol butyrate). The absorbance was measured from the Spectrometer at 415nm. [Shuffle without Lv.1 plasmid.2] was the protein sample from the same batch of the empty SHuffle strain as Tri-PETase constructs. [Shuffle without Lv.1 plasmid.3] was the protein sample from the same batch of the empty SHuffle strain as FAST-PETase constructs. “U”is the amount of activity which releases one micromole of pNP per minute under these assay conditions.  The activity of [Shuffle without Lv.1 plasmid.2] is 1.24E-03 U/mg protein sample. The activity of [Shuffle without Lv.1 plasmid. 3] is 4.44E-03 U/mg protein sample.

In general, all protein constructs showed higher activity towards pNPB comparing to the empty control, which means PETase-silica tag fusion proteins should still be soluble and active. All protein samples containing Tri-PETase or FAST-PETase constructs showed higher fold change of activity towards pNPB compared to the [Dou\_PETase], the old part, under the reaction conditions. The [Car9-linker-Tri\_PETase] showed the best activity when we calculated the fold-change in activity towards pNPB. Both Tri-PETase and FAST-PETase showed increased activity when they fused with silica tags.

##### PETase Immobilization

We incubated the protein sample containing PETase mutants with the silica beads (Celite 545) after activity assessment.. The protein concentration was measured by Bradford assay before and after incubation.

![](https://static.igem.wiki/teams/4390/wiki/results/figure-8-pet.png)

**Figure 23.** The immobilization efficiency of protein samples different PETase constructs after 30 minutes incubation in 4°C. Immobilization efficiency= ([initial protein] - [protein in the washing buffer]) / [initial protein]. The protein concentration in the beginning solution and in the washing buffer was measured by Bradford assay. We load 500ug protein sample to each 20mg Celite545 silica beads for all constructs. [Shuffle without Lv.1 plasmid.2] was the protein sample from the empty SHuffle strain of the same batch (Batch #2) for Tri-PETase. [Shuffle without Lv.1 plasmid.3] was the protein sample from the empty Shuffle strain of the same batch (Batch #3) for FAST-PETase.

From the immobilization efficiency, the protein samples containing [FAST\_PETase-linker-Car9] showed the highest immobilization efficiency over all the constructs (100%). And the protein samples containing [FAST\_PETase-linker-L2NC] showed the lowest immobilization efficiency (11.93%). The Tri-PETase with silica tag shows low immobilization efficiency comparing to the FAST\_PETase with silica tags after we corrected the data with empty control respectively.

The [C-terminal L2NC-tagged FAST-PETase] and [C-terminal L2NC-linker-tagged FAST-PETase] showed the immobilization efficiency (18.65% and 11.93%, respectively), lower than the empty control’s in the same batch. One possible reason might be the positively charged L2NC would interact with negatively charged PETase surface and inhibit the immobilization function.

We observed that the protein samples containing no silica-tag fusion proteins and untagged PETase both showed immobilization behaviour to the silica beads when they were not attached with any silica tags. Two main reasons may cause the results:

1. The container we used to incubate protein with silica beads was 1.5ml Eppendorf tube, and when using it, the certain portion of silica beads always precipitated at the bottom during incubation. The proteins may be trapped in the silica beads precipitated at the bottom. And the unspecific interaction between immobilized PETase and the surrounding proteins may also contribute to the immobilization rate.
2. The surface of the silica support is negatively charged at pH greater than three due to the deprotonated silanol groups. And PETase has a highly polarized surface charge, creating a dipole across the molecule and resulting in an overall isoelectric point (pI) of 9.6. Consequently, there is chance that untagged PETase can immobilized on the silica beads without the facilitation of silica tags.

##### Immobilized PETase activity assessment

We assessed the immobilized PETase activity based on pNPB assay. Same as before, we calculated the fold-change of immobilized protein sample activity over the empty control in the same batch (See Figure 24).

![](https://static.igem.wiki/teams/4390/wiki/results/figure-9-pet.png)

**Figure 24.** The immobilized protein sample activity result based on para-nitrophenol-butyrate (pNPB) assay. The figure presented the fold-change of immobilized protein samples activity over the activity in empty control from the same batch. The fold changes of activity from [Car9-Dou\_PETase] to [Tri\_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.2]. The fold changes of activity from [FAST\_PETase] to [FAST\_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.3]. The reaction system was set up with final volume 1ml in each Eppendorf tube, and the reaction continued for 30 min in 37°C (45 mM Na2HPO4-HCl (pH 7.0) 90 mM NaCl, and 10% (v/v) DMSO; 2mM pNPB-para-nitrophenol butyrate). The absorbance was measured from the Spectrometer at 415nm. [Shuffle without Lv.1 plasmid.2] was the protein sample from the same batch of the empty SHuffle strain as Tri-PETase constructs. [Shuffle without Lv.1 plasmid.3] was the protein sample from the same batch of the empty SHuffle strain as FAST-PETase constructs. “U”is the amount of activity which releases one micromole of pNP per minute under these assay conditions.  The activity of [Shuffle without Lv.1 plasmid.2] is 4.42E-02 U/mg protein sample. The activity of [Shuffle without Lv.1 plasmid. 3] is 1.55 E-02 U/mg protein sample.

The [FAST\_PETase-linker-L2NC] shows the highest activity towards pNPB after immobilizing on the Celite545 (Figure 24) (16.62-fold higher than empty control), but its immobilization rate was the lowest (Figure 25). Referring to the relatively low immobilization efficiency of [FAST\_PETase-linker-L2NC], we assumed that the protein loading on each silica bead should be low. If not, the enzymes may crowd together and inhibit each other’s activity. The activity of all Tri-PETase-silica tag fusion protein was lower than the empty control, and the activity of most FAST-PETase-silica tag fusion protein was at the similar level as the empty control. The data indicated that the protein loading per silica bead should be well defined to maintain the enzyme activity after immobilization.

To assess the reusability of [FAST\_PETase-linker-L2NC], we tested the same sample’s activity again on pNPB assay after washing out the remaining substrate and product from last reaction (See Figure 25).  Consequently, the remaining activity of [FAST\_PETase-linker-L2NC] was 62.96%.

![](https://static.igem.wiki/teams/4390/wiki/results/figure-10-pet.png)

**Figure 25.** The comparison of [FAST\_PETase-linker-L2NC] activity in first and second round of pNPB assay. The reaction system was set up in Eppendorf with final volume 1ml in each Eppendorf tube, and the reaction continued for 30 min in 37°C (45 mM Na2HPO4-HCl (pH 7.0) 90 mM NaCl, and 10% (v/v) DMSO; 2mM pNPB-para-nitrophenol butyrate). The absorbance was measured from the Spectrometer at 415nm.

##### PET biodegradation

We chose the immobilized [FAST\_PETase-linker-L2NC] construct to continue the PET fragment degradation experiment. The heat-treated PET plastic sample was provided by Dr. Joanna Sadler. We increase the pH of reaction system since the product of terephthalic acid (TPA) would lower the pH of reaction environment. We assessed the weight loss of PET plastic fragments after reacting with immobilized PETase.

![](https://static.igem.wiki/teams/4390/wiki/results/figure-11-pet.jpg)

**Figure 26.** The comparison of PET plastic fragment before and after reaction. The fragments circled in red were the same piece of PET fragment. The fragments squared in red were the same piece of PET fragment. The number on the bottom right was the weight of the white tray. The reaction was happened in 100 mM KH2PO4-NaOH buffer (pH 8.0) in 37° C for 2 weeks.

After the reaction, we took out all the PET fragments from the reaction system and dehydrated them in 37°C incubator for 2 days. We observed the fissure in the plastic fragment (red square in Figure 26), and the less irregular edges of the plastic fragment (red circle in Figure 26). The results showed that the immobilized FAST-PETase can slightly degrade the PET plastic. The weight of PET fragment before reaction was 51.8 mg, and after reaction was 51.6mg. There was no significant weight loss of PET fragment, and it may result from multiple factors:

1. The optimized working temperature of FAST-PETase is 50°C, which is 13°C higher than our condition;
2. The amount of [FAST\_PETase-linker-L2NC] loaded on each silica beads is low, at most 107.34µg;
3. Although the immobilized [FAST\_PETase-linker-L2NC] can still react with pNPB, the C-terminus silica tag may interfere the interaction between FAST-PETase and actual PET polymer in reaction. Therefore, we supposed further assessment based on N-terminal tagged FAST-PETase should be conducted with PET sample after optimized immobilization. Considering the higher inhibition effect of N-terminal silica tag (Figure 20), we can try to substitute the N-terminal unstructured peptides (M1 to A27) with the silica tag.
4. Since the PET fragment is insoluble, PETase may react better with it in relatively static environment, instead of continuously shaking. Also, proteins with silica tag may immobilise in the mesopores in the silica beads, and they can’t directly contact with the insoluble PET polymer.

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