Overview
The objective of our project is to find a way to prevent microplastics from continuing to accumulate in the environment, as there are currently no effective solutions to this problem, we decided to develop a biofilm capable of trapping plastic particles. The main objective was to overexpress the Curli fibers which are the main protein component of the biofilm and then add Plastic-specific peptides and a Rubber-binding domain to the Curli fibers.
To achieve our goal, we had to engineer and construct several elements, but also create new protocols that would allow us to verify the identity of our constructs.
In these sections we are going to present how we went through the design-build-test-learn cycles:
Rubber-binding domain
Overview
After discussion with experts, we learned that particles from car tyres can also cause serious damage to aquatic ecosystems (see section Integrated human practices). To tackle this problem we decided to expand our project, by functionalizing our biofilm so that it would be able to capture particles from car tyres.
Design
In literature, we found a protein domain (Als3) that has been shown to bind to rubber.3 As the rubber domain is too big to be directly fused to csgA and exported, we used a different approach. We fused a SpyTag to csgA and from a second plasmid hosted in BL21 E. coli cells we expressed the Rubber-binding protein fused to the SpyCatcher. These cells are lysed and the supernatant is then added to the Curli expressing cell, in this case our working strain PB_002.
Hence, to make this system work, we used two different plasmids: one plasmid contains the genes for the csg domain fused to the Spytag, and the second plasmid contains the genes for the Rubber-binding protein fused to the SpyCatcher.
The plasmid pFM_tag that contains the gene for SpyTag domain was kindly given to us by Roberto Avendaño Vega. The plasmid that contains the genes for the Rubber-binding protein and SpyCatcher was engineered by our team.
Build
We started the cloning process (see section Experiments, Cloning protocol) from the plasmid PN2_013 that we have used as our backbone. We have also ordered from IDT the sequence of the gene als3 that codes for the Rubber-binding protein. The first step was the amplification of the backbone (fragments size: 3198 bp). To do that we designed a pair of primers that would amplify almost all the sequence of PN2_013 except for the lacZ gene, that encodes for a beta-galactosidase, that in our project is not of any interest. With a PCR we have also amplified the als3 gene (fragments size: 1323 bp).
We continued the building process with the Gibson Assembly which joined the two previously amplified fragments to create our engineered plasmid pFM_RB.
The cloning process was concluded with the transformation of the plasmid in E. coli strain NEB5α and plating on the medium LB + Spec50.
To check the correct assembly of the plasmid, we performed a Colony PCR. We verified the junction between the gene encode for the SpyCatcher and als3. For each candidate we obtained a single band and the length of the fragment that we expected (609 kb) (see Figure 4).
This result was confirmed also by the sequencing (see Figure 5).
After confirming the successful building we have transformed the plasmid pFM_RB in the working strain E. coli BL21.
Test
Once we knew that we had obtained the correct plasmid, the next step was to test whether the Rubber-binding protein can be correctly expressed and whether the SpyTag/SpyCatch interaction was working correctly.
As we said in the section before, the Rubber-binding system is composed of two different plasmids that have been transformed into two different working strains. In the strain BL21 we inserted the cloned plasmid called pFM_RB and in our working strain PB_002 we inserted the plasmid pFM_tag.
As the Rubber-binding domain does not have an export tag, the cell is not capable of exporting it. So it is necessary to lyse the BL21 cells. Once the cells are lysed, the Rubber-binding proteins can interact with the Spytag present on the Curli fibers produced by our working strain PB_002 (strain lacking the csg operon).
To test the SpyTag-SpyCatcher interaction, we followed the protocol for Bacterial Lysate SDS-PAGE Gel
(see section Experiments). Briefly the idea of this protocol was to lyse both
strains BL21 and PB_002 and then create three different solutions: The first solution contains BL21 cell lysate
and the Rubber-binding domain-SpyCatcher protein. The second solution contains PB_002 cell lysate and the Curli
fibers modified with the SpyTag. In the last one we would mix the two lysates.
After preparing the solutions we run the proteins on an SDS-PAGE gel. The three overexpressed protein
complexes differ in their molecular weight so we would expect to see three different bands that would
stand out from the background of the cellular proteins.
First, we decided to run the gel on a crude extract hoping for the fact that the proteins will be enough overexpressed to detect the bands. Unfortunately, we could not identify clear bands at the expected sizes (Figure 7).
Learn
Since the proteins were not enough overexpressed to be seen in the crude extract, we decided to redo the process by adding a step of purification that would allow us to isolate our desired proteins.
Second cycle: Test
To improve the result quality of the precedent SDS-PAGE, we decided to add to the protocol Bacterial Lysate SDS-PAGE Gel a
purification step, more precisely a His-tag purification with metal beads (see section Experiments, SDS-PSGE protocol).
At the base of this procedure is the interaction between metal ions and the imidazole ring of histidine.
1
Indeed, our Rubber-domain also contains a His-tag. This purification process enriches the Rubber-binding domain
(SpyCatcher protein + Rubber-binding protein) and the Rubber-binding domain bound to CsgA through the SpyTag protein
from the crude lysate. This process would allow us to hopefully see more clean bands, as the cellular proteins should
not bind to the beads. It is important to underline that this method would not work for the isolation of the CsgA-Spytag
as it does not contain a His-tag, so we used it as a negative control.
From the gel, we still cannot identify clear bands for the proteins we are looking for. We think this result is due to an error made during the His-tag purification. In fact, even after the purification process we still obtained several bands corresponding to cell fragments from the lysis process.
Second cycle: Learn
Due to the lack of time it was not possible to redo the process with the His-tag purification. From the results that we have obtained until now it is possible to conclude that we were able to correctly assemble the plasmid that contains the Rubber-binding domain (Colony PCR and sequencing). If we would have more time, we would repeat the purification to see if we can see SpyTag and SpyCatcher interacting. Afterwards we would test if this biofilm can successfully capture rubber from tyres.
This is a new composite part that we added to the registry:
BBa_K685000
Functionalization of the Curli fibers with Plastic-binding peptides
Overview
In order to improve the plastic binding capacity of the biofilm, we wanted to add Plastic-binding peptides to the Curli fibers.
Design
We identified Plastic-binding peptides from the literature.2 Our aim was to fuse them to the csgA gene via a linker (Figure 10).
For more information check the Design
To clone the DNA sequence coding for the Plastic-binding peptides into our plasmid we first decided to design a short forward primer (PR_010) and a long reverse primer (PR_peptide) containing the linker, the peptide and the overhang with the reverse primer (see Figure 11). Furthermore, we divided our plasmid into two fragments as it was too long for one PCR (~8500 bp).
Build
After having designed our primers, we started the building part. We performed two PCRs. The first PCR consisted of adding the peptides and amplifying one half of the plasmid from csgG to csgA (using PR_003 and PR_peptide). The second PCR amplified the second half of the plasmid from csgC to csgG. As a template we used the plasmid pC3.
Test
To test if the two PCRs yielded fragments of the sizes we expected we performed a gel electrophoresis.
We expected a size of ~2200 bp for fragment 1 and a size of ~5300 bp for fragment 2. As you can see in Figure 12, we did not get the results that we expected. For the backbone, we got two bands and none of them were of the right size. For the fragments, some of them just did not get amplified and some got multiple bands from which one had the right length.
Learn
The PCRs carried out did not yield the expected fragments. Our hypothesis for those results is the following: As the primer PR_pepitde is really long, it has a high chance of forming homodimer, this would explain the smallest bands we got on our gel.
Second cycle: Design
We decided to change our design. We first created a fragment (“template”) containing the linker and the specific peptide. To amplify this part, we designed two primers (PR_041 and PR_peptide_R) and designed more primers to amplify pC3 as backbone (PR_042 and PR_043). The power of this technique compared to our former one is that no primer has an excessive length thus limiting homodimer formation. The primer designed to amplify pC3, the backbone (PR_042 and PR_043) contain a region of overhang with the primers used to amplify the template (PR_041 and PR_peptide_R) in order to close the plasmid during the Gibson Assembly.
Second cycle: build
We did a 1st PCR to amplify the template and a second to amplify the backbone. After having amplified the sequence of interest and the backbone we carried out a Gibson Assembly using pC3.
Second cycle: Test
To test if the template containing the linker and the peptide had been correctly amplified we ran a PCR using primers PR_041 and PR_peptide_R. We were expecting a size of 149 bp which is what we got.
By PCR we also amplified the plasmid pC3 the backbone. After having carried out the Gibson Assembly we
transformed the plasmids in the cloning strain NEB5α.
To check that our sequence contained the specific peptide and the linker we did a Colony PCR using primers
RAV_14 and RAV_16 and ran an electrophoresis. We expected a size of 1036 bp. We got a fragment of the corresponding size.
Unfortunately, the sequencing did not show any traces of the linker nor the peptide. We suspected that the primers we used amplified the csg operon present in NEB5α cells and that we sequenced the genome region. Therefore, we isolated the cloned plasmids and sent the plasmids for sequencing. The results confirmed that the peptide and the linker were not present.
Second cycle: Learn
We hypothesize that the colonies that grow after transformation contain the original pC3 that we used as template during PCR. As the modification is only 149 bp it is impossible to differentiate in our gel the two plasmids. To prevent this from happening again, we should digest our PCRs with Dpn1, an enzyme that digests methylated DNA. The original plasmid in NEB5α is methylated and would be digested, leaving only the amplified non-methylated DNA. Thus, we are sure that all transformants come from the Gibson Assembly.
In summary, we designed and made progress in building a library of plastic-binding peptides. We uploaded the peptides as parts (BBa_K685003, BBa_K685004, BBa_K685005, BBa_K685007, BBa_K685008, BBa_K685010, BBa_K685014, BBa_K685015, BBa_K685020, BBa_K685022, BBa_K685023) and their fusions to csgA as new composite part ( BBa_K685034, BBa_K685035 BBa_K685036, BBa_K685033 BBa_K685038, BBa_K685040, BBa_K685042, BBa_K685037, BBa_K685039, BBa_K685041, BBa_K685043 ). Once built, we would characterize them using the protocol we established to measure plastic capturing Measurement page