iGEM 2024 — Team manchester

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Notebook

Contents

[November](#section1)
[December](#section2)
[January](#section3)
[February](#section4)
[March](#section5)
[April](#section6)
[May](#section7)
[June](#section8)
[July](#section9)
[August](#section10)
[September](#section11)

November

(27 November 2023): Team formation

All members of our team were chosen

(29 November 2023): Our first informal meeting

We met up in a local cafe to get to know each other

December

(6 December 2023): Our first formal meeting

In our first meeting we chose themes that we were most interested in:

* Food and nutrition
* Diagnostics
* Therapeutics
* Personal health/women’s health
* Drugs and drug delivery

We split into small groups to research these themes and come up with a list of ideas.
We also discussed everyone’s skills and interests and their main motivation for joining iGEM and from there gained a better picture of who would be responsible for what aspect of the project.

(13 December 2023): Our second meeting

A delegation from each subteam reported back on their findings and we built upon those ideas. Our PIs then informed us that the subteam model was not a good idea for the early stages, so the subteam model was dissolved, however we continued to build upon the ideas that we had already.

January

January was an exam period, so we were brainstorming ideas on our own and preparing for our exams

February

(14 February 2024): Our third meeting

After researching in individual groups and brainstorming a variety of ideas for our project, we finally decided on what will later be known as "E.Lectrode"!

(21 February 2024): Our first team building

We met up to play the ultimate team building exercises, such as mafia and card games

March

(2 March 2024): Our fourth meeting

We posted recruitment flier to encourage other iGEM teams to collaborate on our project and we have created a possible first draft of our team’s mascot/logo.

Also, we created a link tree to all iGEM socials and we created our email account.

All members of our team were allocated to the following teams: wet lab, dry lab, human practices and wiki development.

(6 March 2024): We met up to take individual and group photos for our social media.

Specific roles were assigned for each team member

Also, we came up with several project ideas: Arachnobacter, Cyber-SPili, Electro-Spili, spiderSENSE, E-rachnid and Silktronics

(13 March 2024): We applied for iGEM mentorship

We had a meeting, where we decided on our project name "spiderSENSE" and chose our villages:

* Therapeutics
* Diagnostics
* Biomanufacturing

(16 March 2024)

Several of our team mebers have attended webinar about Kernel, where we learnt that Kernel is a program for creating plasmids

April

(25 April 2024): First human practices meeting

We met with Robert Meckin, Philip Shapira and Claire Holland from the Manchester Innovation Centre The key points from this meeting are:

* Exploring alternative uses for our nanowires, like using them for brain-machine interfaces, ECG electrodes, or hearing aids, rather than focusing only on prosthetics.
* Before focusing on prosthetics, consult with doctors, clinics, and prosthetic users to identify real needs and potential issues.
* If the product is not cheaper, it needs to offer significant improvements in functionality or user experience to be commercially viable.
* Don’t narrow the focus to prosthetics too early. There may be better applications in other areas like cochlear implants, spinal injuries, or even veterinary uses.
* Expand outreach to include a wider range of contacts in healthcare and research, and work on comparing the conductivity of the nanowires with existing technologies to determine feasibility.

May

May was mainly exam peiod so we mostly did literature search and were brainstorming ideas and team names.

(5 May 2024): Fundraiser

We organized a fundraiser in collaboration with the Latin American society, where we sold baked goods and sweet bags. The event was a success, raising £197, which we donated to the charity Operation Smile as part of our collaboration used to help fund our trips to UK iGEM meetups.

June

(7 - 14 June 2024): Induction week

We started Induction week in the lab to get a basic grip of techniques we will be using in the future like restriction digest, PCR, miniprepping, making and using competent cells, concentration calculations and making up our own reagents for the first time.

July

July was an intense month where we were brainstorming experimental design, experimenting with different plasmids, primers,  E. coli  strains and our protocols in general.

(17 July 2024): Start of the lab experiments

We have finally started our lab experiments. We began with restriction digest and cloning.

(26 July 2024): UK teams iGEM meet up in Warwick

In Warwick, we had an incredible opportunity to meet and collaborate with other UK teams. We presented our poster and enjoyed seeing the innovative work of other teams as they showcased theirs. To top it off, we were thrilled to win both the bingo competition and the Lab Olympics! The experience was not only fun and engaging but also insightful, as we realized that many teams are tackling similar challenges and sharing our journey. It was an inspiring event that brought us closer to the wider scientific community.

August

(7 August 2024): Salford meeting

We arranged a meeting with two clinical prosthetics experts: Professor Washington, who specializes in lower limb prosthetics, and Professor Kirkwood, who works with both lower and upper limb (myoelectric) prosthetics. They found our project concept intriguing, though they acknowledged their limited expertise in biology and myoelectric systems. However, they provided valuable insights:

* The primary challenge with upper limb prosthetics is the excessive weight causing muscle fatigue and the difficulty in coordinating multiple articulations in the hand, elbow, and wrist.
* Other challenges that prosthetic users face are extensive trainings with occupational therapists. Also, heat and friction can cause skin problems like fungal infections, especially in heavy prosthetics.
* We also started to better understand how prosthetics are build and what materials they made of. It was a crucial point for us to understand how we can apply our product in the future
* Prosthetics and therefore our product must meet biocompatible requirements, such as materials must be hypoallergenic, cleanable, and resistant to heat retention to avoid sweating and discomfort. A good material interface is essential for prosthetic comfort and effectiveness.
* Moreover, we got a better understanding what impacts signal transmission in prosthetics: loss of electrode-skin contact, sweat, poorly fitting sockets, and misplacement of electrodes.
* We were recommended to reach out to Reach UK and Alex Chadwell at Newcastle for further research and potential collaboration.

(7 August 2024): Meeting with BAPO

British Association of Prosthetists and Orthotists (BAPO) is dedicated to enhance standards of prosthetic and orthotic care. We reached out to them to gain deeper insights into the experiences of prosthetic users and explore ways to enhance their overall experience. The key points from this meeting are:

* The main barriers for prosthetic use are discomfort, cost, and difficulty in daily use. Also, children prefer not to wear upper limb prosthetics.

September

(7 - 8 September 2024): UK iGEM meetup in Sheffield

At Sheffield, we had the incredible opportunity to lead an educational activity during the university's open day, which earned us the award for the best education activity. We also presented our project to refine it for the upcoming Jamboree and listened to other teams present. Additionally, we attended some captivating talks organized by the Sheffield team. Overall it was a fun and helpful experince for us.

(25 September 2024): The end of lab

As the academic year began, we shifted our focus from completing lab work to concentrating more on write-ups and our studies.

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Engineering

Contents

[Introduction](#section1)
[Iteration 1 - Choice of Chassis Strain](#section2)
[Iteration 2 - Gene Deletion](#section3)
[Iteration 3 - Collagen Binding Assay](#section3)
[References](#references)

Introduction

Type I pili are encoded by the *fim* operon in *Escherichia coli*  and are filamentous structures on the surface of the cell [1]. To prevent interference from the native pili, previous studies that have utilised *E. coli*  as a chassis organism for the heterologous expression of type IV e-pili first created a strain of *E. coli* lacking the *fimA* gene (Ecocyc ID b0530), which encodes for the major structural subunit of type I pili, to serve as the host for further e-pili expression [2]. As our project involved the expression of a heterologous pili monomer in *E. coli*, our first aim was to create a strain of *E. coli* that does not express type I pili to serve as a chassis in further steps of our project.
In the second phase of our project, we aimed to express type IV e-pili modified with a collagen binding peptide in the pili-deficient strain and test for the collagen-binding efficiency of these fusion proteins. Currently, there are numerous experimental procedures available that can determine the binding affinity of a substrate to a protein. However, most of them are very cost ineffective and are not available without specialised equipment (e.g., ELISA, radioligand binding). For this reason, we have decided on using a live cell adhesion assay, which provides an indication whether our recombinant cells adhere to collagen. Additionally, we have adapted a protocol reported in a previous study [3] by substituting crystal violet staining, used to visualise the cells through absorbance measurement, with a fluorescence-based quantification method. Instead of staining the cells, we equipped them with a plasmid which expresses red fluorescent protein (RFP). Our method for testing therefore is a more simplified and sensitive approach.

Iteration 1 - Choice of Chassis Strain

Design:

Initially, we chose *E. coli* NEB10b *ΔfimA* as our final chassis strain because it was previously used by Ueki et al., 2020 [2] for the heterologous expression of type IV e-pili. To build this strain, unlike Ueki et al., 2020, we decided to leverage the CRISPR-Cas12a system designed by Jervis et al., 2021 [4] in order to delete the *fimA* gene of *E. coli* NEB10b.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins, Cas9 and Cas12a (Cpf1), among others, is a genome editing system often used due to its efficiency and ease of use [5]. CRISPR genome editing systems incorporate a gRNA that is integrated into a Cas protein. The gRNA directs the Cas protein to the edit site to produce a double-stranded break (DSB) in the DNA. The system then relies on the cell’s intrinsic homology-directed repair (HDR) mechanism to repair the DSB with provided dDNA [6].
The CRISPR-Cas12a system of Jervis et al., 2021 [4] employs two plasmids: the first plasmid, pSIMcpf1 (Addgene ID: 153034), carries the Cas12a nuclease and the Lambda Red recombination genes. The second plasmid (referred to as pTF) expresses the guide RNA (gRNA) and carries the repair template, composed of two 50-bp homologous arms. Because of its modularity, we only needed to introduce our target gene spacer and donor DNA (dDNA) sequence into the pTF plasmid.
Following the protocol of [4], we designed our NEB10b *fimA* spacer and homology arms. First, the target gene was screened for Cas12 protospacer adjacent motifs (PAM) with the sequence TTTV (where V is A, C, or G). We selected a PAM located in the middle of the gene and used the proceeding 23-bp sequence as *fimA* spacer. Our custom dDNA was composed of the left and right homology arms of the *fimA* gene; the left homology arm was designed to be the 50 bp upstream of the gene’s 5’ end, including the start codon of *fimA*, while the right homology arm was designed to contain 50 bp of the gene’s 3’ end, including the last 9 codons.

![DPlasmid maps of our pTF_fimA plasmid, the pSIMcfp1 plasmid, and the pTF plasmid.](https://static.igem.wiki/teams/5177/engineering-page/fig-1-plasmid-maps-crispr-engineering.webp)

**Figure 1. Plasmid maps of our pTF\_fimA plasmid, the pSIMcfp1 plasmid, and the pTF plasmid.** Diagrams of the pSIMcfp1 plasmid and pTF plasmid were directly sourced from [4]. Our custom *fimA* gene spacer, noted by “fimA spacer 1,” and dDNA sequence (homology arms), labelled “LHA” and “RHA,” were designed to be cloned into the pTF plasmid. We used the pSIMcfp1 plasmid without modification.

Build:

We were kindly provided with the original pSIMcfp1 plasmid and the pTF plasmid (also named pTargettyrRsingle plasmid) used in the original study. We could use the pSIMcfp1 plasmid directly, but the pTF plasmid needed to be tailored to our use. To do so, we chose to design primers (Figure 2B and 2C) to amplify the pTF plasmid by PCR in two parts containing our desired spacers and homology arms and subsequently assemble the two fragments by HiFi DNA assembly into one single pTF\_fimA plasmid (Figure 2A). Simultaneously, we transformed *E. coli* NEB10b cells with plasmid pSIMcfp1.

![Figure 2. Oligonucleotide primers used in the customisation of the pTF plasmid for the deletion of the fimA gene.](https://static.igem.wiki/teams/5177/engineering-page/fig-2-oligonucleotide-primers.webp)

**Figure 2. Oligonucleotide primers used in the customisation of the pTF plasmid for the deletion of the *fimA* gene. [A]**  Overall positioning of the primers on the final plasmid, pTF\_fimA, **[B]** forward and reverse primers to introduce the *fimA* spacer and the left homology arm, **[C]** forward and reverse primers to introduce the right homology arm and amplify the plasmid backbone.

To confirm whether the assembly was successful, we sequenced the resulting plasmid by Sanger sequencing and confirmed that we had obtained the desired pTF\_fimA plasmid. This plasmid was subsequently transformed into the *E. coli* NEB10b cells that already carried plasmid pSIMcfp1, initiating the CRISPR-Cas12a-mediated deletion of *fimA* in *E. coli* NEB10b.

Test:

To confirm whether the CRISPR-Cas12a-mediated deletion of the *fimA* gene was successful, we performed colony PCR of 10 transformants.

![Figure 3. Primers for colony PCR of the NEB10b genome to confirm the deletion of the fimA gene](https://static.igem.wiki/teams/5177/engineering-page/fig-3-colony-pcr.webp)

**Figure 3. Primers for colony PCR of the NEB10b genome to confirm the deletion of the *fimA* gene.** Created with Biorender.com

![Figure 4. Agarose gel electrophoresis results of colony PCR DNA fragments (Day 6 in Gene Deletion Lab Book).](https://static.igem.wiki/teams/5177/engineering-page/fig-4-gel.webp)

**Figure 4. Agarose gel electrophoresis results of colony PCR DNA fragments (Day 6 in Gene Deletion Lab Book).** Lane 1: Ladder, Lanes 2 to 11: colony PCR results for 10 colonies, and Lane 12: Ladder. Lane 1: Ladder, Lanes 2 to 11: colony PCR results for 10 colonies, and Lane 12: Ladder. If the *fimA* gene was successfully removed, a PCR fragment of 692 bp was expected, while an unsuccessful gene deletion would be indicated by a fragment length 1205 bp. As seen, the colony PCR failed for colonies 2, 3, and 10 (lanes 3,4, and 11), while the other 7 colony PCR reactions all yielded the wild type genotype of 1205 bp, meaning that the *fimA* gene deletion was unsuccessful.

As seen in Figure 4, the *fimA* deletion was completely unsuccessful. Therefore, to diagnose the reason behind the failure, we chose to restreak the cells transformed with both the pTF\_fimA and pSIMcfp1 plasmids onto new plates. As the plasmids provided streptomycin and hygromycin resistance, respectively, we restreaked the cells onto LB agar plates containing both antibiotics (LB+Streptomycin+Hygromycin) as well as plates with only one of the antibiotics (LB+Streptomycin and LB+Hygromycin, respectively).

Learn:

Through the results of our diagnostic plates, we discovered that *E. coli* NEB10b had an intrinsic streptomycin resistance, which correlated with why the cells without plasmid pTF\_fimA were able to grow.

![Figure 5. Diagnostic plates from the day before.](https://static.igem.wiki/teams/5177/engineering-page/fig-5-plates.webp)

**Figure 5. Diagnostic plates.** Separate day cultures of cells transformed with both plasmids (labelled as CRISPR A and CRISPR B), as well as cells transformed with only plasmid pSIMcfp1 (A and B respectively) were restreaked onto **[A]** LB+Hygromycin, **[B]** LB+Streptomycin, and **[C]** LB+Streptomycin+Hygromycin.

All restreaks grew on plate [A] as expected, while CRISPR B unexpectedly did not grow on plate [C] even though it should have resistance to both streptomycin and hygromycin. However, the most concerning issue was the growth of cells that were transformed with only the pSIMcfp1 plasmid (A, B) when they were restreaked on both [B] and [C], as the pSIMcfp1 plasmid only provided hygromycin resistance. This growth led us to discover that NEB10b had intrinsic streptomycin resistance.

Faced with this problem, we were presented with two general options: we could either modify plasmid pTF\_fimA and substitute the antibiotic resistance gene for an alternative resistance marker (e.g., *ampR*) for the *smR* gene, or we could research alternative *E. coli* strains to determine whether they had the same *fimA* gene sequence as NEB10b.
We chose the second option, because our plasmids could be directly applied in a strain with the same *fimA* sequence as NEB10b, saving the time of reconstructing plasmid pTF\_fimA. Therefore, we began to investigate various lab strains, notably the *E. coli* NEB5a strain as a potential strain option, considering its close relationship to the NEB10b strain as well as its comparable transformation efficiency.

Iteration 2 - Gene Deletion

Design:

To confirm whether plasmid pTF\_fimA was directly usable in *E. coli* NEB5a, we analysed the NEB5a genome for similar protein sequences as the NEB10b *fimA* gene. We discovered that the NEB5a strain carries a gene labelled *NEB5a\_02280* in its genome (Genbank ID: AOO68850.1) that encodes for the same fimbrial protein as the NEB10b *fimA* gene. This gene also possesses the exact same nucleotide sequence across both strains, meaning that we could immediately begin the *fimA* deletion procedure in NEB5a cells with our plasmids.

![Table Comparision](https://static.igem.wiki/teams/5177/engineering-page/fig-6-table-comparision-of-genes.webp)

**Figure 6: Sequences of NEB10b *fimA* and its corresponding NEB5a counterpart.**

Build:

We transformed the pSIMcfp1 and pTF\_fimA plasmids sequentially into NEB5a following the same protocol we used for the NEB10b cells (See Gene Deletion Lab Book for details).

Test:

We also discovered that our colony PCR primers (Figure 3) bound in the exact same way as they did in the NEB10b genome, meaning that the colony PCR could be performed and results could be interpreted in the same way as for our first iteration.

![Figure 7. Primers for colony PCR of the NEB10b genome to confirm the deletion of the NEB5a_02280 gene.](https://static.igem.wiki/teams/5177/engineering-page/fig-7-colony-pcr.webp)

**Figure 7. Primers for colony PCR of the NEB10b genome to confirm the deletion of the *NEB5a\_02280* gene. Created with Biorender.com**

![Figure 8. Colony PCR results of <i>E. coli</i> NEB5a following the protocol for chromosomal deletion of NEB5a_02280.](https://static.igem.wiki/teams/5177/engineering-page/fig-8-gel.webp)

**Figure 8. Colony PCR results of E. coli NEB5a following the protocol for chromosomal deletion of *NEB5a\_02280.***
**Lane 1:** 1 kb ladder, **Lanes 2 to 11:** colony PCR results for 10 colonies, **Lane 12:** negative control (resuspended cells were substituted with MQ H2O), **Lane 13:** positive control of *E. coli* NEB5a, **Lanes 14 and 15:** 1 kb ladder.

Just like our previous colony PCR, a resulting PCR product of 692 bp was expected if the gene was successfully removed, while an unsuccessful gene deletion would be indicated by a fragment length 1205 bp. As showcased in Figure 8, the gene deletion efficiency was 50% (5/10 colonies screened). For one colony (lane 4 in Figure 2), a mixed genotype could be observed.

Learn:

We successfully deleted *NEB5a\_02280* in the NEB5a genome, but we eventually became aware through genome analysis that NEB10b did not possess the K12 MG1655 *fim* operon and therefore did not have the K12 MG1655 fimA sequence. Rather, the gene sequence normally labelled *sfmA* in K12 MG1655 was labelled as *“fimA”* in the NEB10b strain due to its high protein homology with *fimA*, meaning that the *NEB5a\_02280* gene we deleted was actually the *sfmA* gene of NEB5a instead of its genuine *fimA* gene. This led us to discover that *E. coli* K12 MG1655, and accordingly, the NEB5a strain, possessed multiple chaperone-usher operons homologous to the *fim* operon. The most highly expressed of these homologous operons in standard laboratory conditions was the *sfm* operon [7], which we have incidentally disrupted by removing *sfmA*.
Interestingly, it has also been shown that the removal of any of these chaperone-usher operons provides benefits to cell growth and can potentially increase the biosafety of the strain [8]. Therefore, instead of deleting the *fimA* gene of wild-type NEB5a cells, we determined it would allow us to capitalise on these benefits and also achieve our original goal of preventing native pili expression if we created an *E. coli* NEB5a *ΔsfmAΔfimA* strain by deleting the *fimA* gene of our NEB5a *ΔsfmA* strain.
In order for us to do this, we designed a plasmid with the actual NEB5a *fimA* spacer and homology arms. We named this new plasmid pTF\_fimA and changed the name of the original pTF\_fimA plasmid to **the pTF\_sfmA plasmid** to be consistent (diagrams of Figures 1 and 7 adjusted for this are shown in our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page).

![Figure 9. Proposed pTF_fimA plasmid Map](https://static.igem.wiki/teams/5177/engineering-page/fig-9-plasmid-map.webp)

**Figure 9. Plasmid map for the proposed pTF\_fimA plasmid and a close-up diagram of its fragment cassette.**  Our bespoke 23-bp *fimA* gene spacer, indicated by “fimA spacer,” and dDNA sequence (homology arms), labelled “LHA” and “RHA,” are shown in both diagrams.

We ultimately did not have time to create and use the newly designed pTF\_fimA plasmid (Figure 9) to delete the *fimA* gene in our NEB5a *ΔsfmA* strain. However, we consider that our NEB5a *ΔsfmA* strain was sufficient to serve as our final chassis, as the native fimbriae should only impact the yield of our desired heterologous type IV e-pili and not its expression (which we designed to be controlled by IPTG induction).

Iteration 3 - Collagen Binding Assay

Design:

After creating an *E. coli ΔsfmA* strain (initially thought to be *ΔfimA*) to maximise production of recombinant pili, our plan was to express e-pili with collagen-binding tags. The presence of fimbriae in our strain should not impact the results of the collagen binding assay, as we expected there to be a significant difference of collagen-binding capacity between the fimbriae-expressing cells (*E. coli ΔsfmA*) and the fimbriae-expressing cells that also expressed the e-pili with the collagen-binding tag.
There were multiple options for plasmid construction, and we considered two distinct approaches:

1. A single plasmid containing all genes needed for the type IV pilus assembly machinery and gs*pilA* with the protein-binding tag.
2. Two plasmids: one containing gs*pilA*, including protein-binding tag, and one half of the pili assembly machinery, while the other contained the remaining second half of the assembly machinery.

We decided to use two plasmids to produce the recombinant pili, as the two-plasmid design is more flexible and allows us to change the tags easily. Furthermore, the size of a single plasmid containing all of the genes that code for the assembly machinery would have been over 10000 bp long, resulting in a a decrease in transformation efficiency.

Build:

To build the plasmid system, we selected two compatible BglBrick vectors: pBbA1k and pBbE1c. The modified version of plasmid pBbA1k (hereafter referred to as A1k) carried one half of the assembly machinery, while the modified version of plasmid pBbE1c (hereafter referred to as E1c) contained an unmodified version of gs*pilA* (referred to as E1c-WT), gs*pilA* with a collagen-binding tag (TKKTLRT, named E1c-C1; or LRELHLNNN, named E1c-C2) or a His-tag, as well as the second half of the assembly machinery (see our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page for diagram).
The plasmids have distinct origins of replication and antibiotic resistance markers to ensure uniform replication and avoid plasmid loss during co-transformation. In order for the cells to be used in the adapted collagen-binding assay, we co-transformed these cells with a third plasmid, pBbS1a-RFP (hereafter referred to as S1a), that equipped the cells with the RFP enabling us to determine the cell’s capacity to bind to collagen by measuring RFP fluorescence.

Test:

The following strains were tested for their ability to bind to collagen:- *E. coli ΔsfmA* (negative-control strain)
- *E. coli ΔsfmA* A1k S1a (control strain lacking gs*pilA*)
- *E. coli ΔsfmA* A1k S1a E1c-WT (control strain expressing the unmodified gs*pilA*)
- *E. coli ΔsfmA* A1k S1a E1c-C1 (strain expressing gs*pilA*, modified to encode the collagen-binding tag “TKKTLRT” at its 3’ end)
- *E. coli ΔsfmA* A1k S1a E1c-C2 (strain expressing gs*pilA*, modified to encode the collagen-binding tag “LRELHLNNN” at its 3’ end)
The strains were grown on LB-agar containing the appropriate antibiotics, resuspended in phosphate-buffered saline (PBS), and transferred to wells of 96-well microtitre plates that are coated with rat tail collagen 1 (Thermo Fisher Scientific; A1142803) and regular non-collagen coated plates. After an incubation period the wells were washed to remove cells that had not bound to the well surface. Subsequently, RFP fluorescence was quantified using a plate reader. For a detailed protocol, see [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

![Figure 10. Fluorescence readings of transformed E.coli NEB5a strains.](https://static.igem.wiki/teams/5177/engineering-page/fig-10-graph-1.webp)

**Figure 10. Absolute fluorescence units of the various *E.coli* NEB5a strains.** The first four strains were added to the wells at an OD600 of 2.0. The strain carrying E1c-C2 was added at an OD600 of 0.5. Number of technical replicates: 4.

The fluorescence reading of a control plate with unwashed wells was measured to find out the baseline fluorescence level of the cultures.

**Table 1. Absolute fluorescence units of cultures after the incubation step.** Separate wells of the non-coated plate were not washed and contain the original culture. The first four strains were added to the wells at an OD600 of 2.0. The strain carrying E1c-C2 was added at an OD600 of 0.5. Number of technical replicates: 4.

| 100 μL culture | Average | Stdev |
| --- | --- | --- |
| *ΔsfmA* | 43.0 | 4.0 |
| *ΔsfmA* A1k S1a | 48445.3 | 284.8 |
| *ΔsfmA* A1k S1a E1c-WT | 33558.8 | 399.5 |
| *ΔsfmA* A1k S1a E1c-C1 | 35240.8 | 422.5 |
| *ΔsfmA* A1k S1a E1c-C2 | 6558.3 | 144.0 |

Learn:

The results from the collagen binding assay might suggest that the bacteria generally bind better to the control plate than to the collagen-coated plate. The control strain *ΔsfmA* exhibited a low RFP signal, indicating cell autofluorescence. *E. coli* that was transformed with the plasmid containing the minor pilin and prepilin-peptidase genes, A1k, showed larger fluorescence transmission values in all three of the experiments (Figure 10 and Table 1) compared to the bacteria that supposedly expressed wild-type pili and pili modified with the collagen 1 tag. The fluorescence level of strain *E. coli ΔsfmA* A1k S1a in liquid medium (Table 1) is significantly higher than the level in strains *E. coli ΔsfmA* A1k S1a E1c-WT and  *E. coli ΔsfmA*  A1k S1a E1c-C1. This is most likely the result of a decreased metabolic burden on the cells due to the lack of the third plasmid. The data from Figure 10 shows that *E.coli* transformed with E1c - C2 also lacks the ability to bind to specifically bind to collagen .
Potential causes for these results could be the following:

* Metabolic Burden
As mentioned above, metabolic burden could be a cause for these results.This is the most likely reason, since the data in Table 1 shows this trend in the unwashed control plates. Potential fixes for this could be changing the expression chassis, creating a design with less plasmids or even using the original method from [3], which stained the cells with crystal violet instead of transforming them. This is the most likely reason for the bacteria not binding to collagen, since the data in Table 1 shows this trend.* Collagen Binding Tag
The second reason for the lack of collagen binding could be that the tags modified the structure of the pilin monomer, potentially leading to misshapen or possibly the absence of pili. A way to test this would be to take electron microscope pictures of the cells and check for the presence of pili.* Pili Protein Expression
The GsPilA protein may not have been expressed sufficiently, leading to no pili formation. Thus, it is critical to check for its presence using a Western Blot. Western Blotting the GsPilA with His will test for pili expression. After pili expression is determined, the protein yield can be optimised. This can be remediated using a Design of Experiments (DoEs) aimed to optimise protein yield and expression and increase pili formation.

DoEs is applied statistics that deals with planning, conducting, analysing, and interpreting controlled tests to evaluate the factors that control the value of a parameter or group of parameters. The main goal of this is to optimise the use of factors that affect the yield of pili to maximise protein expression. This can be done through two ways: a full factorial or a fractional factorial. In this case, utilising a full factorial will be optimal as it eliminates ambiguity caused by confounding factors by isolating them across different variables, unlike fractional factorial designs, which may leave certain interactions unresolved.

These are the factors chosen that may affect pili production:

1. Incubation temperature - Newly transcribed recombinant proteins have more time to fold correctly when protein expression occurs at slower rates. Furthermore, correct folding is favoured by the decrease in the concentration of cellular proteins. Reducing incubation temperature may lower protein aggregation as folding of proteins is temperature dependent [9].
2. IPTG Concentration - IPTG is responsible for removing the repressor from the lac operon to induce gene expression [10].
3. Induction Time of cells - Heterologous proteins in *E.coli* require longer times and/or molecular chaperones to fold correctly, thus optimal induction time may be critical in protein folding and formation [11]. After a fixed time of initial incubation on plates (ex. 48 hours, as our cells tended to grow very slowly), cells would be scraped off, resuspended in media, and plated onto new IPTG-containing plates. These plates can then be incubated for various lengths of time to vary induction time.
4. Media Type Carbon Source - Utilising two different carbon sources would demonstrate its role in protein expression, as *E.coli* consumes glucose preferentially over other carbon sources by system regulation and Carbon Catabolite Repression (CCR), which involves a sugar transport system known as phosphoenolpyruvate-phosphotransferase system (PTS system) [12]. When creating M9, the carbon source choice in the protocol used was glycerol, choosing glucose as a carbon source might facilitate high protein integrity as glycerol is linked to faulty protein folding [13].

Factors that we can change to maximise protein expression:

Incubation Temperature {2 levels}

* 16 ℃
* 30 ℃

Inducer Concentration (IPTG) {2 levels}

* 50 μM
* 250 μM

Induction Time of Cells (Depends on Temperature and IPTG Concentration) {2 levels}

* 24 hrs / Day 1
* 48 hrs / Day 2

Media Type {2 levels}

* M9 Composition

+ Glucose
+ Glycerol

Prospective Plan:
We will first do a trial run to see the feasibility of the experiment and adjust it accordingly to ensure it works.
We do a full factorial unreplicated design of 40 trials to screen the best possible combination. See the math below.
Factor 1 = 2 levels, Factor 2 = 2 levels , Factor 3 = 2 levels, Factors 4 = 2 levels

Find out how many trials we need to run!

2 x 2 x 2 x 2 =

Check Answer

After knowing which combination produces the most protein, we will test again to verify our initial conclusion and replicate the experiment/method 3 different times with our chosen factors.
This full factorial analysis will help find the optimal parameters of the factors chosen to increase protein expression in E.coli.

References

* Schilling, J.D., Mulvey, M.A., Hultgren, S.J. Structure and Function of *Escherichia coli* Type 1 Pili: New Insight into the Pathogenesis of Urinary Tract Infections. *The Journal of Infectious Diseases* [Internet]. 2001 Mar;183(s1):S36–40. Available from: <https://academic.oup.com/jid/article/183/Supplement_1/S36/2191070>
* Ueki T., Walker, D.J.F., Woodard, T.L., Nevin, K.P., Nonnenmann, S.S., Lovley, D.R. An *Escherichia coli* Chassis for Production of Electrically Conductive Protein Nanowires. *ACS Synthetic Biology*. 2020 Mar 3;9(3):647–54. Available from: <https://pubs.acs.org/doi/10.1021/acssynbio.9b00506>
* Grenier, D. Collagen-binding activity of *Prevotella intermedia* measured by a microtitre plate adherence assay. *Microbiology*. 1996 Jun 1;142(6):1537–41. Available from: <https://pubmed.ncbi.nlm.nih.gov/8704994/>
* Jervis, A.J., Hanko, E.K.R., Dunstan, M.S., Robinson, C.J., Takano, E., Scrutton, N.S. A plasmid toolset for CRISPR‐mediated genome editing and CRISPRi gene regulation in *Escherichia coli*. *Microbial Biotechnology*. 2021 Mar 12;14(3):1120–9. Available from: <https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.13780>
* Asmamaw, M., Zawdie, B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing. *Biologics : Targets & Therapy* [Internet]. 2021 Aug 21;15(1):353–61. Available from: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388126/>
* Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., Zhang, F. Genome engineering using the CRISPR-Cas9 system. *Nature Protocols*. 2013 Oct 24;8(11):2281–308. Available from: <https://www.nature.com/articles/nprot.2013.143>
* Korea, C.G., Badouraly, R., Prevost, M.C., Ghigo, J.M., Beloin, C. *Escherichia coli* K-12 possesses multiple cryptic but functional chaperone-usher fimbriae with distinct surface specificities. *Environmental Microbiology*. 2010 Mar 23;12(7):1957–77. Available from: <https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2010.02202.x>
* Qiao, J., Tan, X., Ren, H., Wu, Z., Hu, X., Wang, X. Construction of an *Escherichia coli* Strain Lacking Fimbriae by Deleting 64 Genes and Its Application for Efficient Production of Poly(3-Hydroxybutyrate) and l -Threonine. *Applied and Environmental Microbiology* [Internet]. 2021 May 26;87(12). Available from: <https://journals.asm.org/doi/10.1128/aem.00381-21>
* Rosano, G.L., Ceccarelli, E.A. Recombinant protein expression in *Escherichia coli*: Advances and challenges. *Frontiers in Microbiology*. 2014 Apr 17;5(172). Available from: <https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2014.00172/full>
* Simas, R.G., Pessoa, A., Long, P.F. Mechanistic aspects of IPTG (isopropylthio-β-galactoside) transport across the cytoplasmic membrane of *Escherichia coli* – a rate limiting step in the induction of recombinant protein expression. *Journal of Industrial Microbiology & Biotechnology*. 2023 Oct 17;50(1). Available from: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10639102/>
* Francis, D.M., Page, R. Strategies to optimize protein expression in *E. coli*. *Current Protocols in Protein Science* [Internet]. 2010 Aug 1;Chapter 5:Unit 5.24.1-29. Available from: <https://www.ncbi.nlm.nih.gov/pubmed/20814932>
* Terol, G.L., Gallego-Jara, J., Martínez R.A.S., Díaz, M.C., de Diego Puente, T. Engineering protein production by rationally choosing a carbon and nitrogen source using *E. coli* BL21 acetate metabolism knockout strains. *Microbial Cell Factories*. 2019 Sep 4;18(1). Available from: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724240/>
* Chiang, C.J., Ho, Y.J., Hu, M.C., Chao, Y.P. Rewiring of glycerol metabolism in *Escherichia coli* for effective production of recombinant proteins. *Biotechnology for Biofuels*. 2020 Dec;13(1). Available from: <https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-01848-z>

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Experiments

## Gene Deletion Lab Book:

If the PDF does not work, please visit:
[*Gene Deletion Lab Book*](https://static.igem.wiki/teams/5177/experiments-page/final-correct-gene-deletion-lab-book-compressed.pdf).

## Vector Construction Lab Book:

If the PDF does not work, please visit:
*Vector Construction Lab Book*.

## Protein Purification Lab Book:

If the PDF does not work, please visit:
[*Protein Purification Lab Book*](https://static.igem.wiki/teams/5177/experiments-page/protein-purification-lab-book.pdf).

## Primers for Vector Construction:

If the PDF does not work, please visit:
[*Primers for Vector Construction*](https://static.igem.wiki/teams/5177/experiments-page/primers.pdf).

## All Protocols Followed:

If the PDF does not work, please visit:
[*All Protocols Followed*](https://static.igem.wiki/teams/5177/experiments-page/protocols.pdf).

{% endblock %}

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{% block title %}Results{% endblock %}
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Results

Contents

[Aims](#aims)
[Creation of Custom Bacterial Chassis](#section1)
[Vector Construction](#section2)
[Collagen Binding](#section3)
[Project Achievements](#section4)
[Future Directions](#section5)
[References](#references)

Aims

The aim of E.lectrode is to produce pili derived from *Geobacter sulfurreducens* with a skin-binding tag at the C-terminus of the pilus to enhance its functionality.
This involves the following key steps:

1. **[Creation of Fimbriae-Deficient *E. coli* Chassis](#section1):** Build a strain of Δ*fimA E. coli* NEB5a by performing gene deletion utilising a CRISPR-Cas12a system.
2. **[Vector Construction](#section2):** Construct two vectors using Gibson assembly.
3. **[Expression and Functionality Assessment](#section3):** Express modified e-pili by co-transformation of vectors into the Δ*fimA* strain, and test the binding affinity of the pili to collagen.

See [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) for more detail.

Creation of Fimbriae-Deficient *E. coli* Chassis

CRISPR-Cas12a-mediated Deletion of a Gene Encoding for a Fimbrial Protein

The *fimA* gene (Ecocyc ID b0530) encodes the major type 1 pili structural subunit protein in *Escherichia coli (E. coli)* NEB5a.
As our project involves the expression of a heterologous pili monomer in *E. coli* NEB5a,
we needed to prevent the expression of the native type 1 pili to ensure maximal production of our desired type IV e-pili,
and therefore we aimed to chromosomally delete *fimA* in *E. coli* NEB5a.
To create this strain, we sought to employ a CRISPR-Cas12a system [1] that can be easily adapted to be used for chromosomal gene deletion in *E. coli*.
This system employs two plasmids: the first plasmid, pSIMcpf1 (Addgene ID: 153034), carries the Cas nuclease and the Lambda Red recombination genes.
The second plasmid (referred to as pTF) expresses the guide RNA (gRNA) and carries the repair template, composed of two 50-bp homologous arms.
Because of its modularity, we only needed to introduce our target gene spacer and donor DNA (dDNA) sequence into the pTF plasmid,
and could then carry out the gene deletion process with this modified plasmid along with the pSIMcfp1 plasmid.

However, we eventually discovered through genome analysis that the gene we deleted was instead the *sfmA* gene
of this strain and that a labeling error in the databases we used (NCBI and Biocyc) led us to design our CRISPR-Cas12a system around this gene (see [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) page for details).
While this was not originally the plan,
we quickly realised that deleting both *sfmA* and the true *fimA* would actually be beneficial,
resulting in increased growth in minimal environments [2], which is beneficial because we will be expressing the e-pili with M9.
However, we did not have time to complete the double deletion.

![Primers for modification of pTF plasmid to form pTF_sfmA](https://static.igem.wiki/teams/5177/results/figure-1.webp)

**Figure 1. Oligonucleotide primers used in the customisation of the pTF plasmid for the custom gene deletion. (A)**  Overall positioning of the primers on the final plasmid, pTF\_sfmA, **(B)** forward and reverse primers to introduce the *sfmA* spacer and the left homology arm, **(C)** forward and reverse primers to introduce the right homology arm and amplify the plasmid backbone.

![pTF_sfmA, pSIMcfp1, pTF plasmid maps](https://static.igem.wiki/teams/5177/results/figure-2.webp)

**Figure 2. Plasmid maps of the pTF\_sfmA plasmid, the pSIMcfp1 plasmid, and the pTF plasmid.** Diagrams of the pSIMcfp1 plasmid and pTF plasmid were directly sourced from [1], and the pTF\_sfmA plasmid map was created with BioRender.com. pTF\_sfmA was used in conjunction with pSIMcfp1 in our deletion of the NEB5a *sfmA* gene.

We were able to design a new fragment cassette with a gene spacer and dDNA for the actual NEB5a *fimA* gene,
but we had too little time to experimentally delete the actual *fimA* gene in our NEB5a *ΔsfmA* strain.

![Plasmid map of pTF_fimA and zoomed-in view of fragment cassette.](https://static.igem.wiki/teams/5177/results/figure-3.webp)

**Figure 3. Plasmid map for the proposed pTF\_fimA plasmid and a close-up diagram of its fragment cassette.** Our custom 23-bp *fimA* gene spacer, noted by “fimA spacer (1),” and dDNA sequence (homology arms), labelled “LHA” and “RHA,” are shown in both diagrams. Created with Biorender.com

Although it is reported to be cryptic,
we do not think deleting *sfmA* was a wasted effort,
as it is a putative fimbrial protein-encoding gene nonetheless and its deletion would further aid the growth rate of our host in minimal media [2].
Thus, this phase of our project was a success, achieving a 50% gene deletion efficacy as shown by our colony PCR (Figure 5).

![Plasmid map of pTF_fimA and zoomed-in view of fragment cassette.](https://static.igem.wiki/teams/5177/results/figure-4.webp)

**Figure 4. Primers for colony PCR to confirm the deletion of the *sfmA* gene.** Created with Biorender.com

![Gel electrophoresis results of the colony PCR fragments following sfmA deletion.](https://static.igem.wiki/teams/5177/results/figure-5.webp)

**Figure 5. Colony PCR results of *E. coli* NEB5a following the protocol for chromosomal deletion of *sfmA*.** **Lane 1:** 1 kb ladder, **Lanes 2 to 11:** colony PCR results for 10 colonies, **Lane 12:** negative control (resuspended cells were substituted with MQ H2O), **Lane 13:** positive control of *E. coli* NEB5a, **Lanes 13 and 14:** 1 kb ladder.

Eventually, through multiple rounds of plasmid curing,
we successfully procured a *ΔsfmA* strain of *E. coli* NEB5a (see Gene Deletion Lab Book in our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page for details).
After we discovered we would also need to delete the *fimA* gene to fully remove all native fimbriae formation,
we originally aimed to repeat the gene deletion process with our newly designed
pTF\_fimA plasmid and produce an *E. coli* NEB5a *ΔsfmAΔfimA* strain to be used as the chassis of our e-pili production,
but due to time constraints,
we chose to directly use our *ΔsfmA* strain in the later stages of our project.

Vector Construction

Workflow

The proposed workflow for creating the pilus expression vectors involves several key steps.
The type IV pilus assembly genes will be amplified from the *E. coli* K12 genome using PCR (Figure 6).
These amplified fragments, along with a synthetic gene block containing the *Geobacter sulfurreducens* pilus (Gs*pilA*),
will then be divided into two groups and assembled into two separate pBbE1k sub-plasmids using Gibson assembly (Figure 7).
The genes in the first sub-plasmid will include Gs*pilA*, *hofB-hofC*, and *hofM-hofN-hofO-hofP-hofQ*,
while the second sub-plasmid will contain *ppdA-ppdB-ygdB-ppdC* and *gspO*.
Finally, these fragments will be amplified from the sub-plasmids and assembled into the final pBbE1c vector through Gibson assembly,
resulting in a complete plasmid capable of expressing the pilus.

![Type IV pilus assembly gene fragments](https://static.igem.wiki/teams/5177/results/primer-v3.webp)

**Figure 6. Type IV pilus assembly gene fragments from K12.**
The four gene fragments of the type IV pilus assembly machinery to be amplified from the K12 genome,
with Gibson assembly overhangs added for subsequent cloning steps.
The fragments are: (A) *hofB-hofC*, (B) *hofM-hofN-hofO-hofP-hofQ*, (C) *gspO*, and (D) *ppdA-ppdB-ygdB-ppdC*.
Created with BioRender.com

![OG plan](https://static.igem.wiki/teams/5177/results/subclonal-5.webp)

**Figure 7. Pili expression vectors.**
The gene fragments Gs*pilA*, *hofB-hofC*, and *hofM-hofN-hofO-hofP-hofQ* will be assembled into one pBbE1k sub-plasmid (A),
while the fragments *ppdA-ppdB-ygdB-ppdC* and *gspO* will be assembled into another pBbE1k sub-plasmid (B).
The two resulting fragments from these sub-plasmids will then be combined and assembled into the final plasmid using Gibson assembly (C).
Created with BioRender.com

Our original vector design (Figure 7), where a single promoter controlled the expression of the entire pilus assembly system, raised some concerns.
Redesigning the primers to insert a promoter in front of each operon was not feasible due to time constraints.
However, because our initial plan involved subcloning, the overhangs are compatible with other BglBrick vectors.
This compatibility allowed us to select a different BglBrick vector for one of the sub-plasmids, enabling co-transformation of the vectors.
As a result, we could place a promoter in front of two operons (Figure 8). This approach provided a practical solution,
improving the regulation of gene expression without requiring extensive redesign.

![Co-transformation vectors](https://static.igem.wiki/teams/5177/results/final-plasmid-5.webp)

**Figure 8. Modified pilus assembly vector design.**
The sub-plasmid backbones were changed from pBbE1k to pBbA1k (A) and pBbE1c (B) to optimise the system,
enabling direct co-transformation of the sub-plasmids without the need for prior assembly into a single plasmid.
This new design incorporates two promoters for enhanced gene expression control. Created with BioRender.com

PCR Amplification of Type IV Pilus Assembly Machinery from K12 Genome and Plasmid Preparation for Gibson Assembly

The BglBrick vectors pBbE1c-RFP and pBbE1k-RFP were streaked out for single colonies.
Red/pink colonies were observed, indicating successful expression of the RFP reporter gene (Figure 9).
A random colony was chosen from one of the pBbE1c-RFP plates and the pBbE1k plate to inoculate liquid cultures and placed in the 37℃ incubator overnight.

![BglBrick vectors with RFP on plate](https://static.igem.wiki/teams/5177/results/plates-678.webp)

**Figure 9. Streaked out cells with BglBrick vectors for single colonies for overnight cultures.**
Cells containing the BglBrick vectors pBbE1c-RFP (A and B) and pBbE1k-RFP (C) were streaked out onto LB plates containing either chloramphenicol or kanamycin.

The pBbA1k vector was streaked out for single colonies and red/pink colonies were observed,
indicating successful expression of the RFP reporter gene (Figure 10).
A random colony from the plate was selected to inoculate a liquid culture.

![A1k-RFP](https://static.igem.wiki/teams/5177/results/a1k-plate.webp)

**Figure 10. Streaked out cells with pBbA1k vectors for single colonies.**
Cells containing the BglBrick vector pBbA1k were streaked out onto LB plates supplemented with kanamycin.

After the miniprep, we used a NanoDrop to measure the DNA concentration of the samples (Table 1),
allowing us to calculate the volume of vector needed for the restriction digest with BamHI and NdeI.

**Table 1. NanoDrop results after minipreparation of vectors from overnight cultures.**

| Vector | Concentration (ng/μl) |
| --- | --- |
| pBbE1c | 179.5 |
| pBbE1k | 118.9 |

The digested vectors and PCR products containing the amplified type IV pilus assembly system gene fragments from K12 were loaded onto a 1% agarose gel and ran at 130V for 30 min.
The results were largely as expected, except that no band was observed for pBbA1a (Figure 11).
This sample, provided by an advisor, was intended to be used in place of pBbA1k if the digest had worked.
However, due to the absence of a band, we proceeded with the original plan to use pBbA1k.

Additionally, no band was observed for the pBbA1a sample.
This sample, provided by an advisor, was intended to be used in place of pBbA1k if the digest had worked.
However, due to the absence of a band, we proceeded with the original plan to use pBbA1k.

![Gel after amplification of machinery](https://static.igem.wiki/teams/5177/results/gel-1-2.webp)

**Figure 11. Size purification of digested BglBrick vectors and PCR products.**
The samples loaded were: lane 1, molecular weight marker (NEB 1 kb ladder); lane 2, blank; lane 3, pBbA1a (3572 bp);
lane 4, pBbE1c (3317 bp); lane 5, pBbA1k (3526 bp); lane 6, *hofB-hofC* (2642 bp); lane 7, *hofM-hofN-hofO-hofP-hofQ* (3354 bp);
lane 8, *ppdA-ppdB-ygdB-ppdC* (1802 bp); lane 9, *gspO* (743 bp); lane 10, molecular weight marker (NEB 1 kb ladder).

The pBbA1k-RFP vectors were miniprepped from the two overnight cultures and a NanoDrop was conducted (Table 2).

**Table 2. NanoDrop results after minipreparation of pBbA1k from overnight cultures.**

| Vector | Concentration (ng/μl) |
| --- | --- |
| pBbA1k-1 | 74.6 |
| pBbA1k-2 | 85.6 |

The pBbA1k vectors were digested with BamHI and NdeI.
The digested vectors were loaded onto a 1% agarose gel and electrophoresed at 130V for 30 min.
The gel shows that the pBbA1k vectors were digested as expected (Figure 12).
Both the pBbE1c vector and pBbA1k vector were digested and purified to be assembled with Gs*pilA* and the type IV pilus assembly system by Gibson assembly.

![Size purification of pBbA1k](https://static.igem.wiki/teams/5177/results/gel-2.webp)

**Figure 12. Size purification of pBbA1k BglBrick vector.**
The two samples were digested with BamHI and NdeI. The expected fragment length is 3513 bp.

PCR Amplification of Gs*pilA* with His and Collagen-Binding Tags

After designing the primers to add tags to Gs*pilA* by PCR, we attempted to simulate the assembly using SnapGene but encountered issues.
The problem arose with the assembly of Gs*pilA* and *hofB*.
The original forward primer for the *hofB-hofC* fragment had an overhang that was compatible with Gs*pilA* without tags.
After consulting with an advisor, we determined we could just shorten the original primer.
Since Gs*pilA* was synthesised with Gibson overhangs, we do not need to engineer a Gibson overhang into the hofB-hofC forward primer (Figure 13).

![Modified hofB-hofC primer](https://static.igem.wiki/teams/5177/results/modified-primer-3.webp)

**Figure 13. Modified hofB-hofC\_fwd primer.**
The original hofB-hofC\_fwd primer amplified the *hofB-hofC* fragment, resulting in incompatible Gibson overhangs.
The modified primer is 20 bp shorter than the original primer, which removes the engineered Gibson overhang, and is now compatible.
Created with Biorender.com.

PCR was conducted to amplify *hofB-hofC* using the modified primer and to incorporate the following tags at the C-terminus of the GsPilA protein:
a His tag and two different collagen-binding peptides, TKKTLRT (C1) and LRELHLNNN (C2).
The PCR products were then loaded onto a 1% agarose gel and run at 130V for 30 min.
As shown in Figure 14, bands corresponding to *hofB-hofC* and Gs*pilA* with a His tag are present.
The band for Gs*pilA*-His migrated below the lowest band of the DNA ladder, suggesting the need to use a 100 bp ladder for accurate identification.
However, no bands were observed for Gs*pilA* with either of the collagen-binding peptides.
This issue might be due to the long overhangs of the primers relative to the annealing regions.
Using a different polymerase or increasing the primer concentration might improve amplification efficiency under these conditions.

![Tags](https://static.igem.wiki/teams/5177/results/gel-3.webp)

**Figure 14. Agarose gel electrophoresis of PCR products amplifying *hofB-hofC* and Gs*pilA* constructs.**
The samples loaded were: lane 1, molecular weight marker (NEB 1 kb ladder);
lane 2, *hofB-hofC* gene fragment (2642 bp); lane 3, Gs*pilA* with His tag (272 bp, highlighted in the red box);
lane 3, Gs*pilA* with collagen-binding peptide TKKLRT (275 bp); lane 5, Gs*pilA* with collagen-binding peptide LRELHLNNN (281 bp).

The PCR to add collagen-binding peptides to Gs*pilA* was repeated using the PCRBIO HS VeriFi™ polymerase.
The PCR products were analysed on a 1% agarose gel at 130V for 30 min (Figure 15).
The bands appeared streaky, making it challenging to determine the precise cutting locations,
therefore, we excised both bands from each sample to ensure comprehensive recovery.
For improved resolution and more accurate differentiation between the bands,
we will use a molecular weight marker tailored for small DNA fragments in future experiments.

![Repeat collagen gel](https://static.igem.wiki/teams/5177/results/gel-4.webp)

**Figure 15. Gs*pilA* with collagen-binding peptides.**
The samples loaded were: lane 1, molecular weight marker (NEB 1 kb ladder);
lane 3, Gs*pilA* with collagen TKKTLRT (275 bp); lane 5, Gs*pilA* with collagen LRELHLNNN (281 bp).
The bands believed to contain Gs*pilA* with the collagen-binding peptides are highlighted in the green box,
while the bands excised as a precaution are marked in the red box.

DNA Purification

The PCR products were excised from the agarose gel and purified using the Zymoclean™ Gel DNA Recovery Kit.
Following purification, the samples were quantified using a NanoDrop spectrophotometer or Quibit fluorometer.
The digested vectors yielded relatively low concentrations. These values, though on the lower side,
are consistent with the expected outcomes for large plasmid backbones after gel extractions (Table 3).

The absence of peaks at 260 nm for pBbE1c, pBbA1k-1, and pBb1k-2 were observed, which could be attributed to several factors,
including potential issues with the gel extraction process, degradation of the DNA, or low DNA concentration.
To confirm the presence of DNA, we used a Qubit fluorometer, which provides more sensitive and accurate quantification (Table 3).

**Table 3. Comparison of NanoDrop and Qubit results after gel extraction of type IV pilus assembly system gene fragments and digested vectors.**
*N/A* indicates that the concentration was not measured by Qubit as a peak at 260 nm was observed.

| Gene | Concentration (ng/μl) from NanoDrop | Concentration (ng/μl) from Qubit |
| --- | --- | --- |
| Gs*pilA* with His tag | 49.6 | N/A |
| *hofB-hofC* | 66.1 | 16.6 |
| *hofB-hofC* using modified primer | 16.5 | N/A |
| *hofM-hofN-hofO-hofP-hofQ* | 50.8 | 66.0 |
| *ppdA-ppdB-ydgB-ppdC* | 78.2 | 119 |
| *gspO* | 56.4 | 83.7 |
| pBbE1c | 25.6 | 38.0 |
| pBbE1k | 38.4 | Not needed, as we are not using this vector. |
| pBbA1k-1 | 10.7 | 12.5 |
| pBbA1k-2 | 19.2 | 22.4 |

The DNA fragments with the collagen-binding tags were purified from the gel using the Zymoclean™ Gel DNA Recovery Kit and subsequently quantified with a Qubit fluorometer (Table 4).

**Table 4. Qubit fluorometer values for Gs*pilA* with collagen.**

| Collagen | Concentration (ng/μl) |
| --- | --- |
| C1 | 30.6 |
| C1.2 | 32.6 |
| C2 | 19.2 |
| C2.2 | 18.7 |

Transformation of Assembled Constructs

The digested pBbE1c vector was assembled with Gs*pilA*-His, *hofB-hofC*, and *hofM-hofN-hofO-hofP-hofQ*,
while the digested pBbA1k vector was assembled with *ppdA-ppdB-ygdB-ppdC* and *gspO* using Gibson assembly.
The resulting constructs were transformed into NEB 5-alpha cells plated onto LB agar containing the appropriate antibiotics.
The plates were incubated overnight at 37℃ (Figure 16). The negative control plate for pBbE1c, which lacked an insert,
had more colonies than the experimental plate. This suggests that the control plate likely contains cells with re-ligated vector backbones.
In contrast, the pBbA1k construct plate had significantly more colonies, indicating successful Gibson assembly of the plasmid.

![Gibson plates](https://static.igem.wiki/teams/5177/results/gibson-plates.webp)

**Figure 16. Transformation of NEB 5-alpha cells with assembled constructs.**
(A) Negative control plate (left): pBbE1c assembled without *hofM-hofN-hofO-hofP-hofQ*.
Experimental plate (right): pBbE1c assembled with all three inserts. Colonies are highlighted with red circles.
(B) Negative control plate (left): pBbA1k assembled without *ppdA-ppdB-ygdB-ppdC*.
Experimental plate (right): pBbA1k assembled with both inserts.

The digested pBbE1c vector was assembled with either Gs*pilA*-C1 or Gs*pilA*-C2, along with *hofB-hofC* and *hofM-hofN-hofO-hofP-hofQ*.
The resulting constructs were transformed into NEB 5-alpha cells plated onto LB agar containing the appropriate antibiotics.
The plates were incubated overnight at 37℃ (Figure 17). The control plates (Figure 17A and 17B, left) had no colonies, while the experimental plates showed colony growth.
This suggests that the transformations were successful only when the complete assembly was present (Fig 17A and 17B, right).
On the C1.2 experimental plate (Figure 17C, left), the presence of colonies implies that the gel fragments might have contained the correct PCR product,
or there may have been an issue with the assembly process leading to unintended DNA incorporation.
The absence of colonies of the C2.2 experimental plate (Figure 17C, right) indicates the Gibson assembly failed, as expected, since it lacked the gs*pilA* insert.

![Collagen gibson plates](https://static.igem.wiki/teams/5177/results/collagen-gibson-plates.webp)

**Figure 17. NEB 5-alpha cells transformed with pBbE1c assembled with Gs*pilA* containing the collagen-binding peptides.**
(A) Negative control for C1 (left): pBbE1c assembled without *hofM-hofN-hofO-hofP-hofQ*.
Experimental plate (right): pBbE1c assembled with Gs*pilA* containing the C1 collagen-binding peptide along with the other two inserts.
(B) Negative control plate for C2 (left): pBbE1c assembled without *hofM-hofN-hofP-hofQ*.
Experimental plate (right): pBbE1c assembled with Gs*pilA* containing the C2 collagen-binding peptide along with the other two inserts.
(C) Experimental plates: pBbE1c assembled with C1.2 (left) and pBbE1c assembled with C2.2 (right).

Verification of pBbE1c Vector Construction Containing His Tag

To verify the construction of the pBbE1c vector, colony PCR was performed on all four colonies from the plate.
The PCR products were then analysed by running them on a 1% agarose gel at 130V for 50 min.
As shown in Figure 18, only one of the colonies produced a band, indicating the presence of the desired construct.

![Colony PCR](https://static.igem.wiki/teams/5177/results/gel-5.webp)

**Figure 18. Colony PCR results for pBbE1c vector construction.**
Gel electrophoresis of PCR products from four colonies,
with only one colony producing the expected 6168 bp fragment (highlighted in a red box).

The only colony from the pBbE1c plate that produced the expected fragment in the colony PCR was used to inoculate a liquid culture.
The vectors were then miniprepped from the overnight culture and quantified with a Qubit fluorometer, resulting in a concentration of 562 ng/μl.

The remaining cells containing the assembled pBbE1c construct, were plated onto LB plates supplemented with chloramphenicol.
The vectors were then purified from the overnight cultures using a miniprep procedure and quantified with a Qubit fluorometer (Table 5).
Both pBbE1c samples were sent off for full plasmid Nanopore sequencing.

**Table 5. Qubit quantification results of miniprepped assembled pBbE1c-His vectors.**

| Vector | Concentration (ng/μl) |
| --- | --- |
| pBbE1c-1 | 642 |
| pBbE1c-2 | 549 |

Verification of pBbE1c Vector Construction Containing Collagen-Binding Tags

To set up overnight cultures, ten random colonies were selected from both the pBbE1c\_Gs*pilA*-C1 and pBbE1c\_Gs*pilA*-C2 plates.
A total of 20 overnight cultures were prepared, miniprepped, and quantified.
The pBbE1C vectors assembled with Gs*pilA*-C1 were quantified with a Qubit fluorometer,
while the pBbE1c vectors assembled with Gs*pilA*-C2 were quantified with a NanoDrop spectrophotometer (Table 6).

**Table 6. Qubit and NanoDrop results for assembled vectors with collagen-binding peptides.**
(Left) Qubit results for assembled pBbE1c vectors with Gs*pilA*-C1, *hofB-hofC*, and *hofM-hofN-hofO-hofP-hofQ*.
(Right) NanoDrop results for assembled pBbE1c vectors with Gs*pilA*-C2, *hofB-hofC*, and *hofM-hofN-hofP-hofQ*.

| Vector sample with C1 | Concentration (ng/μl) | Vector sample with C2 | Concentration (ng/μl) |
| --- | --- | --- | --- |
| 1 | 196 | 1 | 256.2 |
| 2 | 255 | 2 | 215.1 |
| 3 | 250 | 3 | 268.4 |
| 4 | 232 | 4 | 246.6 |
| 5 | 195 | 5 | 232.6 |
| 6 | 190 | 6 | 311.0 |
| 7 | 114 | 7 | 193.8 |
| 8 | 192 | 8 | 142.0 |
| 9 | 204 | 9 | 148.9 |
| 10 | 222 | 10 | 254.6 |

All 20 samples of the pBbE1c vectors containing the collagen-binding peptides were loaded onto a 1% agarose gel and run at 130V for 30 min.
The results were as expected, with all samples displaying three bands (Figure 19).
However, there are variations in the brightness of the bands, which can be due to variations in DNA concentration.
Since all samples were successfully verified by digestion,
we selected the two samples with the highest concentration for each collagen-binding peptide to send for full plasmid Nanopore sequencing.

![Collagen digest verification](https://static.igem.wiki/teams/5177/results/gel-8.webp)

**Figure 19. Digest verification of pBbE1c vectors with both collagen-binding peptides.**
The samples loaded were: lane 1, molecular weight marker (NEB 1 kb ladder);
lanes 2-11, vectors containing C1; lane 12, molecular weight marker; lanes 13-22, vectors containing C2.
The expected fragment sizes for the digested vector with peptide C1 are 4460 bp, 2952 bp, and 2082 bp,
while the expected fragment sizes for the digested vector with peptide C2 are 4466 bp, 2952 bp, and 2082 bp.

Verification of pBbA1k Vector Construction

Six random colonies from the pBbA1k plate were used to inoculate liquid cultures.
The vectors were then miniprepped from the overnight cultures and quantified with a Qubit fluorometer (Table 7).

**Table 7. Qubit quantification results of miniprepped assembled pBbA1k vectors.**

| Sample | Concentration (ng/μl) |
| --- | --- |
| 1 | 37.2 |
| 2 | 18.1 |
| 3 | 21.0 |
| 4 | 18.4 |
| 5 | 45.3 |
| 6 | 63.1 |

To verify the construction of the pBbA1k vectors, a restriction digest was performed using BamHI and NdeI.
The digested vectors were run on a 1% agarose gel at 130V for 30 min.
The results were as expected, revealing two distinct bands at 2455 bp and 3513 bp (Figure 20).

![Digest verification of A1k](https://static.igem.wiki/teams/5177/results/gel-6.webp)

**Figure 20. Gel electrophoresis results of pBbA1k construct digestion with BamHI and NdeI.**
The samples loaded were: lane 1, molecular weight marker (NEB 1 kb ladder);
lanes 2-8, digested pBbA1k constructs from samples 1-6.

Due to limited sample availability for both digestion and plasmid sequencing,
0.5 μl of the overnight culture from each pBbA1k construct was plated onto LB plates supplemented with kanamycin.
The vectors were then purified from the overnight cultures using a miniprep procedure and quantified with a Qubit fluorometer (Table 8).
The pBbA1k constructs with the highest concentration were sent off for full plasmid Nanopore sequencing.

**Table 8. Qubit results of the assembled vectors after minipreparation.**

| Vector | Concentration (ng/μl) |
| --- | --- |
| pBbA1k-1 | 38.5 |
| pBbA1k-2 | 44.0 |
| pBbA1k-3 | 37.8 |
| pBbA1k-4 | 39.6 |
| pBbA1k-5 | 39.4 |
| pBbA1k-6 | 33.7 |
| pBbE1c-1 | 642 |
| pBbE1c-2 | 549 |

Correction of Misassembled Vectors Following Sequencing Verification

All plasmids were sent for full plasmid Nanopore sequencing.
When the results came back, the only vector that had assembled correctly was the pBbA1k vector containing ppdA-ppdB-ygdB-ppdC and gspO.
In contrast, the pBbE1c vectors containing the various binding tags with the rest of the type IV pilus assembly system had misassembled.
The vectors with the His tag and C1 contained a similar unexpected insertion between the end of the Gs*pilA* gene and the tag.
Meanwhile, vectors containing C2 showed a large deletion in the Gs*pilA* gene.
These findings confirmed the presence of a design flaw (see the [Cloning Lab Book](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for more detail).

To correct the pBbE1c vectors with the His tag, we designed primers that amplified the vector in two fragments,
removing the insertion, and then reassembled the vectors using Gibson assembly.
As we got the sequencing results for the vectors containing the collagen-binding peptide after confirming the correct assembly of the pBbE1c vectors with the His tag,
we decided to use the His-tagged vector as a template to assemble the vectors with the collagen-binding peptides in a similar manner to correct the His-tagged vector.
We also designed a primer to remove the tag and produce WT pili to serve as a baseline for conductivity testing.

The two samples of the pBbE1c vectors with the His tag sent for sequencing were both amplified by PCR and run on a 1% agarose gel at 130V for 30 min (Figure 21).
The gel appeared successful, except for one missed well. Fortunately, we had two samples expected to yield the same results.
We excised the bands and purified the DNA using the Zymoclean™ Gel DNA Recovery Kit.
The purified DNA was then assembled using Gibson assembly to generate the plasmid.

![Fixing his](https://static.igem.wiki/teams/5177/results/gel-9-3.webp)

**Figure 21. Gel electrophoresis analysis of pBbE1c vectors with His tags amplified by PCR.**
The samples loaded were: lane 1, molecular weight marker (NEB 1 kb ladder);
lane 2, pBbE1c-His sample 1 amplified with primers iGEM18 and iGEM19;
lane 3, pBbE1c-His sample 1 amplified with primers 20 and 21;
lane 4, pBbE1c-His sample 2 amplified with primers iGEM18 and iGEM19; lane 5, pBbE1c-His sample 2 amplified with primers 20 and 21.
The fragments are highlighted in red boxes, and the expected fragments sizes are labelled.

The assembled vectors were transformed into NEB 5-alpha cells and plated onto LB agar plates supplemented with chloramphenicol (Figure 22).
The growth of the plate suggests successful assembly of the vectors.
However, a negative control was not included, making it difficult to rule out the possibility of background growth.

![Plate of cells transformed with fixed his](https://static.igem.wiki/teams/5177/results/corrected-his-plates.webp)

**Figure 22. NEB 5-alpha cells transformed with the corrected pBbE1c-His vectors.**
The transformed cells were plated onto LB plates supplemented with chloramphenicol and grown overnight.

To set up overnight liquid cultures, three random colonies were selected from the plate.
The plasmids were then isolated using the QIAprep Spin Miniprep Kit and quantified using a Qubit fluorometer (Table 9).

**Table 9. Qubit concentrations of the corrected, miniprepped pBbE1c-His vectors.**

| Sample | Concentration (ng/μl) |
| --- | --- |
| 2 | 151 |
| 3 | 89.5 |

The vectors were digested with BamHI and run on a 1% agarose gel at 130V for 30 min (Figure 23).
Both samples showed the expected three bands, indicating that the vectors likely assembled correctly.
As a result, the vectors were sent for Sanger sequencing.

![Digest verification of fixed his](https://static.igem.wiki/teams/5177/results/gel-10.webp)

**Figure 23. Gel electrophoresis analysis of pBbE1c-His vectors digested with BamHI.**
The samples loaded were: lane 1, molecular weight marker (NEB 1 kb ladder); lane 2, pBbE1c-His sample 1; lane 3, pBbE1c-His sample 2.

To assemble the vectors containing the collagen-binding peptides and vectors without the His tag, we adapted the corrected pBbE1c-His vector.
The amplified fragments were run on a 1% agarose gel at 130V for 30 min (Figure 24).
The gel showed clear single bands for each sample, indicating successful amplification.

![Making vectors with collagen](https://static.igem.wiki/teams/5177/results/gel-11.webp)

**Figure 24. Gel electrophoresis analysis of fragments amplified from pBbE1c-His.**
The samples loaded were: lane 1, molecular weight marker (NEB 1 kb ladder);
lane 2, pBbE1c-C1 fragment 1 (4443 bp); lane 3, pBbE1c-C1 fragment 2 (5111 bp);
lane 4, pBbE1c-C2 fragment 1 (4449 bp); lane 5, pBbE1c-C2 fragment 2 (5114 bp);
lane 6, vector without tag fragment 1 (4443 bp); lane 7 vector without tag fragment 2 (5090 bp);
lane 8, water (negative control); lane 9, molecular weight marker (NEB 1 kb ladder).

The fragments were assembled using Gibson assembly to produce the plasmids.
The plasmids were then transformed into NEB 5-alpha cells and plated onto LB agar supplemented with chloramphenicol (Figure 25).
Colonies can be seen on all three plates, indicating successful assembly.
However, a negative control was not included, making it difficult to rule out the possibility of background growth.

![Transformed cells with assembled collagen and WT](https://static.igem.wiki/teams/5177/results/fixed-collagen-and-wt-plate.webp)

**Figure 25. Transformed NEB 5-alpha cells with vectors adapted from pBbE1c-His.**
(A) pBbE1c with C1; (B) pBbE1c with C2; (C) pBbE1c without tags.

From each plate, three random colonies were selected to set up overnight cultures.
The plasmids were purified using the QIAprep Spin Miniprep Kit and quantified with a Qubit fluorometer (Table 10).
Samples 2 and 3 of the pBbE1c-C2 vector showed notably lower concentrations compared to the other samples.
This may be due to factors, such as suboptimal bacterial growth, plasmid instability, or inefficiencies during the purification process.

**Table 10. Qubit results for the assembled vectors with the collagen-binding peptide and vectors without tags.**

| Sample | Concentration (ng/μl) |
| --- | --- |
| pBbE1c-C1 (1) | 406 |
| pBbE1c-C1 (2) | 582 |
| pBbE1c-C1 (3) | 408 |
| pBbE1c-C2 (1) | 321 |
| pBbE1c-C2 (2) | 177 |
| pBbE1c-C2 (3) | 187 |
| pBbE1c-WT (1) | 336 |
| pBbE1c-WT (2) | 487 |
| pBbE1c-WT (3) | 252 |

To verify the assembly of the vectors, they were digested with BamHI, and the resulting digests were analysed on a 1% agarose gel at 130V for 30 min.
If the vectors had assembled correctly, each sample would show three distinct bands.
This pattern was observed in the pBbE1c-C1 vector samples, and in one of the pBbE1c-WT samples.
In contrast, the other samples displayed a single band, resembling the band of the undigested vector.
The digest was repeated using a shorter incubation time and the high-efficiency NEB BamHI-HF enzyme, but similar results were obtained (Figure 26).
This confirmed that the issue does not lie in enzyme efficiency or DNA degradation, but rather suggests an incorrect assembly of the plasmids.

![Transformed cells with assembled collagen and WT](https://static.igem.wiki/teams/5177/results/gel-12-2.webp)

**Figure 26. Digest verification of pBbE1c vectors containing Gs*pilA* with C1, C2, and WT.**
The samples loaded were: lane 1, MW marker (NEB 1 kb ladder); lanes 2-4, C1 samples 1-3;
lane 5, MW marker; lanes 6-8, C2 samples 1-3; lane 9, MW marker; lanes 10-12, WT samples 1-3;
lane 13, MW marker; lane 14, negative control with BamHI and Cutsmart buffer;
lane 15, water (negative control); lane 16, C1 sample 1 without BamHI (positive control); lane 17 MW marker.

All three C1 samples, C2 sample 2, and WT sample 2 were sent for Sanger sequencing.
The main objective was to verify the end of Gs*pilA* to ensure the correct tag has been added or that the WT sequence is present, with perfect alignment.
While there are some point mutations elsewhere, we have previously checked the plasmid with Nanopore sequencing,
so these mutations might be due to limitations of Sanger sequencing.
For more information on the sequencing results, see [Cloning Lab Book](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

Expression and Functionality Assessment: Collagen Binding

The following strains were constructed and tested for their ability to bind to collagen:
E. coli *ΔsfmA* (negative-control strain)
E. coli *ΔsfmA* A1k S1a (control strain lacking Gs*pilA*)
E. coli *ΔsfmA* A1k S1a E1c-WT (control strain expressing the unmodified Gs*pilA*)
E. coli *ΔsfmA* A1k S1a E1c-C1 (strain expressing Gs*pilA*, modified to encode the collagen-binding tag “TKKTLRT” at its 3’ end)
E. coli *ΔsfmA* A1k S1a E1c-C2 (strain expressing Gs*pilA*, modified to encode the collagen-binding tag “LRELHLNNN” at its 3’ end)
The strains were grown on LB-agar containing the appropriate antibiotics, resuspended in phosphate-buffered saline (PBS), and transferred to wells of 96-well microtitre plates that are coated with rat tail collagen 1 (Thermo Fisher Scientific ; A1142803) and regular non-collagen coated plates. After an incubation period the wells were washed to remove cells that had not bound to the well surface. Subsequently, RFP fluorescence was quantified using a plate reader. For a detailed protocol, see [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

![Collagen Binding Assay Results Graph](https://static.igem.wiki/teams/5177/results/ass-ay-results.webp)

**Figure 27. Absolute fluorescence units of the various *E.coli* NEB5a strains.** The first four strains were added to the wells at an OD600 of 2.0. The strain carrying E1c-C2 was added at an OD600 of 0.5. Number of technical replicates: 4.

The fluorescence reading of a control plate with unwashed wells was measured to find out the baseline fluorescence level of the cultures.

**Table 11. Absolute fluorescence units of cultures after the incubation step.** Separate wells of the non-coated plate were not washed and contain the original culture. The first four strains were added to the wells at an OD600 of 2.0. The strain carrying E1c-C2 was added at an OD600 of 0.5. Number of technical replicates: 4.

| 100 μL culture | Average | stdev |
| --- | --- | --- |
| *ΔsfmA* | 43.0 | 4.0 |
| *ΔsfmA* A1k S1a | 48445.3 | 284.8 |
| *ΔsfmA* A1k S1a E1c-WT | 33558.8 | 399.5 |
| *ΔsfmA* A1k S1a E1c-C1 | 35240.8 | 422.5 |
| *ΔsfmA* A1k S1a E1c-C2 | 6558.3 | 144.0 |

As seen from Figure 27, the results from the collagen binding assay might suggest that the bacteria generally bind better to the control plate than to the collagen-coated plate. We have various theories of why this is so; see our [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) page for more detail.

Project Achievements

Project Successes

* Successfully deleted the *sfmA* gene from *E. coli* NEB 5-alpha.
* Constructed vectors for pili expression.
* Co-transformed the constructed pili expression vectors into *ΔsmfA* *E. coli* NEB 5-alpha.

Project Challenges

* [Gene deletion accuracy](#gene-deletion-accuracy): A labelling error in databases led to the unintentional deletion of *sfmA* gene instead of the intended  gene.
* [Design flaw](#correction): The seqeuncing results revealed that the pili expression vectors was misassembled due to incorrect design. This misassembly resulted in additional time spent fixing the vectors before proceeding.

Future Directions

Please see our [Implementation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D) page for the potential ways our project can be utilised in the future.

References

* Jervis AJ, Hanko EKR, Dunstan MS, Robinson CJ, Takano E, Scrutton NS. A plasmid toolset for CRISPR‐mediated genome editing and CRISPRi gene regulation in Escherichia coli. Microbial Biotechnology. 2021 Mar 12;14(3):1120–9. Available from: <https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.13780>
* Qiao J, Tan X, Ren H, Wu Z, Hu X, Wang X. Construction of an Escherichia coli Strain Lacking Fimbriae by Deleting 64 Genes and Its Application for Efficient Production of Poly(3-Hydroxybutyrate) and L-Threonine. Applied and Environmental Microbiology [Internet]. 2021 May 26 [cited 2024 Apr 25];87(12). Available from: <https://journals.asm.org/doi/10.1128/aem.00381-21>

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