Engineering Success



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:

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:

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.

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:

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.



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