iGEM 2019 — Team Western_Canada

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# Team:Western Canada/Notebook

Western Canada

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  [Notebook](https://2019.igem.org/Team%3AWestern_Canada/Notebook)
* [Dry Lab](https://2019.igem.org/Team%3AWestern_Canada/Dry_Lab)

  [Model](https://2019.igem.org/Team%3AWestern_Canada/Model)
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  [Demonstrate](https://2019.igem.org/Team%3AWestern_Canada/Demonstrate)
[Safety](https://2019.igem.org/Team%3AWestern_Canada/Safety)
* [Human Practices](https://2019.igem.org/Team%3AWestern_Canada/Human_Practices)
* [Attributions](https://2019.igem.org/Team%3AWestern_Canada/Attributions)
* [Judging](https://igem.org/2019_Judging_Form?team=Western_Canada)
* [Collaborations](https://2019.igem.org/Team%3AWestern_Canada/Collaborations)
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# Notebook

To download and view our complete lab notebook, please  [click here.](https://static.igem.org/mediawiki/2019/8/86/T--Western_Canada--Western_iGEM_Notebook_2019.pdf)

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# Team:Western Canada/Experiments

Western Canada

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* [Dry Lab](https://2019.igem.org/Team%3AWestern_Canada/Dry_Lab)

  [Model](https://2019.igem.org/Team%3AWestern_Canada/Model)
  [Structure Prediction](https://2019.igem.org/Team%3AWestern_Canada/Dry_Lab_structures)
  [Demonstrate](https://2019.igem.org/Team%3AWestern_Canada/Demonstrate)
[Safety](https://2019.igem.org/Team%3AWestern_Canada/Safety)
* [Human Practices](https://2019.igem.org/Team%3AWestern_Canada/Human_Practices)
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* [Judging](https://igem.org/2019_Judging_Form?team=Western_Canada)
* [Collaborations](https://2019.igem.org/Team%3AWestern_Canada/Collaborations)
* [HP Education and Engagement](https://2019.igem.org/Team%3AWestern_Canada/Public_Engagement)

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# Experiments

### *Aim 1: Design and assembly of parts into plasmid backbone*

All parts will be ordered from Twist Bioscience as synthetic DNA blocks. Primers with extensions corresponding to regions flanking the insertion site on the shuttle vector pAGE 2.0 (**Table 1**) will be ordered from Integrated DNA Technologies.

**Table 1.** Primers to amplify each part for insertion into the pAGE 2.0 vector plasmid by homology cloning.

|  |  |  |
| --- | --- | --- |
| **Part** | **Forward primer** | **Reverse primer** |
| CsgA:SpyCatcher | iGEM1\_F: CTGTTTCTCCATACCCGTTTTTTGTTTAACTTTAAGAAGG | iGEM1\_Ralt: CAAAATTATTTCTAGCCCAAAATCAATGATGATGATGATGATGAATATGAGC |
| Laccase:SpyTag-B | iGEM5\_F: GGCTGTAATGCCCGTTCGCTC | iGEM2\_R: CAAAATTATTTCTAGCCCAAAATCAATGATGATGATGATGATGC |
| Cutinase:SpyTag | iGEM2\_F: CTGTTTCTCCATACCCGTTTTGACGGCG | iGEM2\_R: CAAAATTATTTCTAGCCCAAAATCAATGATGATGATGATGATGC |
| Laccase:SpyTag-A | iGEM2\_F: CTGTTTCTCCATACCCGTTTTGACGGCG | iGEM5\_R: GGGCATTACAGCCATTGAGCAAAC |
| SpyTag:Laccase-A | iGEM2\_F: CTGTTTCTCCATACCCGTTTTGACGGCG | iGEM4\_R: ATCGCCAAACCAGCCCACG |
| SpyTag:Laccase-B | iGEM4\_F: CTGGTTTGGCGATACGTTGCTG | iGEM2\_Ralt: CAAAATTATTTCTAGCCCAAAATCAATGATGATGATGATGATGTACCGTAAACCC |
| SpyCatcher:CsgA | iGEM1\_F: CTGTTTCTCCATACCCGTTTTTTGTTTAACTTTAAGAAGG | iGEM1\_R: CAAAATTATTTCTAGCCCAAAATCAGTACTGATGAGC |
| SpyTag:Cutinase | iGEM2\_F: CTGTTTCTCCATACCCGTTTTGACGGCG | iGEM3\_R: CAAAATTATTTCTAGCCCAAAATCAATGATGATGATGATGATGGGC |

The vector pAGE 2.0 will be amplified as three overlapping fragments (A, B, and C) with the primers in (**Table 2**). This plasmid contains a chloramphenicol resistance marker for selection in *E. coli*and a histidine biosynthesis complementation gene for selection in the histidine-auxotrophic yeast strain *S. cerevisiae* VL6-48.

**Table 2.** Primers to amplify the pAGE 2.0 vector plasmid in three fragments.

|  |  |  |
| --- | --- | --- |
| **Plasmid Fragment** | **Forward primer** | **Reverse primer** |
| A | BK806F: TTTTGGGCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG | BK573R: GCCAGCCCAGCGGCGAGGGCAACCAGCTCGACTATATTACCCTGTTATCCCTAGCGTAAC |
| B | BK574F: CGCTCCGGCCTGCCTCAATCTCGTTGGCCATGACCGAGCAACGACTGCACCGGATGGTTC | BK574R: GAGAATATTCAGGCCAGTTATGCTTTCTGGCCTGTAACAAAGGACATTAAGTAAAGACAG |
| C | BK575F: CATTCTTAAGGAACTTGAAAAGCCAGCACCCTGATGCGACCTCGTTTTAGTCTACGTTTA | BK805R: AAACGGGTATGGAGAAACAGTAGAGAGTTGCGATAAAAAG |

### *Aim 2: Assaying protein expression and efficiency of enzyme export*

To assay the export of enzymes to the media, western blotting will be used. Transformed bacteria will be grown O/N to OD600=1 in LB-CHL and cultures will be centrifuged to separate the pellet and the supernatant. Supernatant will be treated with trichloroacetic acid, the obtained precipitate, containing the secreted proteins, will be washed with acetone and resuspended in SDS sample buffer. The *E. coli* cells will be lysed using SDS-lysis buffer. SDS-PAGE will be performed on the *E. coli* lysate and supernatant samples, and the proteins will be transferred to a nitrocellulose membrane. Membranes will be blotted with an anti-FLAG 1o antibody and a fluorochrome-conjugated 2o antibody. If western blot results indicate no protein export but presence of the protein in the cell lysate, the secretion constructs will be re-designed to change the relative location of SpyCatcher and the enzyme.

### *Aim 3: Assaying enzymatic activity of the exported enzyme*

The enzymatic activity of secreted cutinase will be determined using an assay based on the cleavage of 4-methylumbelliferyl butyrate (MUB). *E. coli* O/N cultures grown to OD600=1.0 will be centrifuged and the supernatant will be added to 96 well-plate. MUB in phosphate-citrate buffer will be then added to initiate the reaction, and the fluorescence of cleavage product will be measured using a plate reader. The activity of the secreted laccase will be assayed in the similar way but using oxidation of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and LB medium supplemented with 0.1 mM CuSO4.

### *Aim 4: Assaying curli fiber production and growing a composite biofilm*

To assay whether the SpyTag-fused curli fibers are produced successfully, a biofilm growth assay adapted from Uhlich *et al.*, Botyanszki *et al.*, and Chapman *et al.*, will be used1,2,3. *E. coli* MC 4100 ΔcsgA, transformed with the csgA-SpyTag, construct will be first grown in LB- CHL O/N. Bacterial cultures will be centrifuged and the supernatant will be aspirated. Bacterial pellets will be then re-suspended in a nutrient-poor YESCA-CHL medium and grown for 24-48 hours at 25 oC to allow curli fibers to form. Bacterial cultures will then be centrifuged, supernatant will be aspirated, and pellets will be re-suspended in PBS buffer. Expression of curli fibers will be quantified using Congo Red staining and by measuring absorbance at 490 nm, as described by Botyanszki *et al*1.

To form a composite biofilm between the MC 4100 ΔcsgA strain transformed with a csgA-SpyTag construct and MC1000 strain transformed with the secretion-SpyCatcher-enzyme construct, both strains will be inoculated in separate LB-CHL cultures and grown O/N. Two cultures will then be combined, mixed, pelleted and re-suspended in YESCA medium. The obtained combined culture will then be aliquoted to wells on a flat-bottom 24-well cell culture plate and a biofilm will be grown at 25 oC, with gentle shaking. Prolonged growth in a well-plate will allow biofilm to adhere to the plastic and achieve high density.

### *Aim 5: Assaying activity of curli-immobilized enzymes*

The supernatant will be decanted from the wells and the biofilms will be washed with PBS buffer to remove all remaining growth media. This step will also remove all enzymes that have been secreted, but not attached to the curli fibers. The buffer will then be mixed with either MUB or ABTS, depending on which enzyme is being assayed, and added to the wells. After the reaction is complete, the buffer will be removed from the wells, transferred to a different well plate, and the concentration of the reaction products will be determined using a plate reader.

### *Aim 6: Assaying degradation of an environmental pollutant using the composite catalytic biofilm*

As a final step to confirm the feasibility of removing environmental pollutants using the proposed approach, we will assay degradation of 17β-estradiol and diclofenac. Both of those compounds are regarded as emerging environmental pollutants and have been previously shown to be degraded by laccase4. 17β-estradiol and diclofenac will be dissolved in an aqueous buffer of approximately neutral pH over a range of concentrations, with some particulate and organic matter, to approximate the conditions of untreated sewage. This solution will be added to the catalytic biofilm, as described in Aim 5, and incubated for varying amounts of time. Degradation of 17β-estradiol and diclofenac will be monitored using HPLC4.

### *References*

1. Yousra Turki, Y. *et al.* Biofilms in bioremediation and wastewater treatment: characterization of bacterial community structure and diversity during seasons in municipal wastewater treatment process. *Environ. Sci. Pollut. Res.* **24,**  3519–3530 (2017).
2. Hammer, N. D., Schmidt, J. C. & Chapman, M. R. The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization. *Proc. Natl. Acad. Sci. U. S. A.*  **104,**  12494–9 (2007).
3. Lloret, L. *et al.*  Laccase-catalyzed degradation of anti-inflammatories and estrogens. *Biochem. Eng. J.* **51,**  124–131 (2010).
4. Osma, J. F., Toca-Herrera, J. L. & Rodríguez-Couto, S. Cost analysis in laccase production. *J. Environ. Manage.* **92,**  2907–2912 (2011).

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# Team:Western Canada/Results

Western Canada

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  [Notebook](https://2019.igem.org/Team%3AWestern_Canada/Notebook)
* [Dry Lab](https://2019.igem.org/Team%3AWestern_Canada/Dry_Lab)

  [Model](https://2019.igem.org/Team%3AWestern_Canada/Model)
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  [Demonstrate](https://2019.igem.org/Team%3AWestern_Canada/Demonstrate)
[Safety](https://2019.igem.org/Team%3AWestern_Canada/Safety)
* [Human Practices](https://2019.igem.org/Team%3AWestern_Canada/Human_Practices)
* [Attributions](https://2019.igem.org/Team%3AWestern_Canada/Attributions)
* [Judging](https://igem.org/2019_Judging_Form?team=Western_Canada)
* [Collaborations](https://2019.igem.org/Team%3AWestern_Canada/Collaborations)
* [HP Education and Engagement](https://2019.igem.org/Team%3AWestern_Canada/Public_Engagement)

<center>Results</center>

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# Results

### *Preparation of all fragments for assembly*

For Gibson assembly as well as TAR-cloning (yeast assembly), good-quality fragments for all backbone parts as well as synthetic inserts needed to be generated with homology between adjacent fragments (**Figure 1**). To do this, the synthetic DNA parts received from Twist Bioscience were amplified by PCR to have homologous ends compatible the pAGE 2.0 insertion site. In addition, the pAGE 2.0 plasmid was amplified in three overlapping fragments.

![Responsive image](https://static.igem.org/mediawiki/2019/e/e3/T--Western_Canada--results1.jpg)

**Figure 1. Agarose gel electrophoresis of purified PCR amplicons for all fragments.** 1 µL of the purified and pooled PCR products for each fragment was run on a 1.4-% agarose gel at 120 V for 40 minutes.

### *Homology cloning*

Although we were unable to yield *E. coli* transformant colonies from two attempts at the Gibson assembly approach to constructing whole plasmids from the purified fragments, we were successful with our second attempt at yeast assembly (**Figure 2, Table 1**).

![Responsive image](https://static.igem.org/mediawiki/2019/4/4c/T--Western_Canada--results22.jpg)

**Figure 2. Results from Gibson (top) and yeast (bottom) assembly after transformation of *E. coli* and *S. cerevisiae*, respectively.** Top panel: DNA from each Gibson assembly reaction was transformed by electroporation (2500 V) into *E. coli* by mixing 20 µL of reaction product with 200 µL of electrocompetent *E. coli* EPI300 and spreading 500 µL of recovered cells onto LB 1.5% agar plates supplemented with 30 µg/mL chloramphenicol. Plates were incubated for 24 hours at 37°C. Bottom panel: 20 µL of equimolar fragment mixtures for each construct were transformed into *S. cerevisiae* spheroplasts using polyethylene glycol (PEG) and 500 the cell-DNA mixture was suspended in each of two tubes containing 8 mL of molten 1M-sorbitol 2%-agar yeast media lacking histidine, poured into empty Petri dishes, and incubated for three days at 30°C.

**Table 1. *S. cerevisiae* colony counts after yeast assembly.**

|  |  |  |
| --- | --- | --- |
|  | **Plate 1** | **Plate 2** |
| SpyTage:Laccase | 64 | 66 |
| SpyCatcher:CsgA | 93 | 96 |
| CsgA:SpyCatcher | 207 | 151 |
| Laccase:SpyTag | 81 | 78 |
| SpyTag:Cutinase | 107 | 128 |
| Cutinase:SpyTag | 72 | 75 |

### *Validation of assembled plasmids*

Plasmid DNA isolated from the pooled yeast assembly colonies was successfully transformed into *E. coli* EPI300 (**Table 2**) for cloning, diagnostic PCR, and restriction digest assays.

![Responsive image](https://static.igem.org/mediawiki/2019/7/70/T--Western_Canada--results8.jpg)

**Figure 3. *E. coli* colonies transformed with DNA isolated from pooled yeast colonies.** Left panel: 1 µL of DNA isolated from pooled yeast colonies was transformed by electroporation (2500 V) into 40 µL of electrocompetent *E. coli* EPI300. 700 µL of recovered cells were spread onto LB plates supplemented with 30 µg/mL chloramphenicol and incubated for 24 hours at 30°C. Right panel: 20 colonies from each transformation were selected and streaked onto fresh LB plates supplemented with 30 µg/mL chloramphenicol and incubated for 24 hours at 30°C.

**Table 2. *E. coli* colony counts after electroporation.**

|  |  |
| --- | --- |
|  | **Count** |
| SpyTage:Laccase | 123 |
| SpyCatcher:CsgA | 136 |
| CsgA:SpyCatcher | 135 |
| Laccase:SpyTag | 534 |
| SpyTag:Cutinase | 117 |
| Cutinase:SpyTag | 83 |

To identify successfully-assembled plasmid clones, we performed colony PCR using lysate from individual *E. coli* colonies for each construct. The pAGE 2.0 vector has a pair of primers flanking the insertion site, which is the junction between fragments C and A (**Figure 4**). Performing PCR with these primers yields an amplicon sized either 214 bp if no insertion is present or 214 + insertion size if there is an insertion present (**Figure 5**). For reference, pAGE 2.0 with no insert was used as template DNA for PCR using these primers (+).

![Responsive image](https://static.igem.org/mediawiki/2019/8/87/T--Western_Canada--results4.jpg)

**Figure 4. Annotation map of the insertion site in pAGE 2.0.** Figure was generated using Geneious version 2019.2, created by Biomatters (Available from https://www.geneious.com).

![Responsive image](https://static.igem.org/mediawiki/2019/4/44/T--Western_Canada--results3.jpg)

**Figure 5. Gel electrophoresis of colony PCR products from lysed *E. coli* cells from colonies containing assembled plasmids.** 1 µL of the products from the colony PCR of the plasmid insertion site (**Figure 4**) was run on a 1.4-% agarose gel at 120 V for 40 minutes. PCR products yielding electrophoresis bands of the right size are identified with green circles.

From these colony PCR results, we selected putatively-positive clones (circled in green) and inoculated cells from the corresponding colonies into selective liquid media to grow overnight. In the morning, 1 mL from each saturated culture was transferred to a 250-mL culture flask containing 50 mL of selective liquid media supplemented with arabinose to induce expression through the pBAD promoter. Our constructs were designed such that pBAD drives an operon encoding the inserted open reading frame as well as monomeric red fluorescent protein (mRFP), both of which are preceded by a ribosome binding site (RBS). The diluted & induced cultures were placed for six hours at 37°C in an incubator set to shake at 225 rpm.

The cultures were transferred to 50-mL Falcon tubes and centrifuged at 3000 rcf for 25 minutes at 4°C. The supernatants were discarded and the pelleted cells were observed for the presence of red fluorescence (**Figure 6**). Since our inserts are polycistronic with, and upstream of, mRFP, red fluorescence indicate that our inserts are at least being transcribed. Since the same RBS sequence was used for both open reading frames, we can also reasonably predict that fluorescing cells are producing our synthetic proteins.

![Responsive image](https://static.igem.org/mediawiki/2019/0/0c/T--Western_Canada--results6.jpg)

**Figure 6. Pelleted cells from induced cultures of *E. coli* containing putatively-positive clones of each plasmid.** Cultures were centrifuged for 25 minutes at 3000 rcf at 4°C after inducing for 6 hours with 98 µg/mL arabinose and 225-rpm shaking at 37°C.

Based on the presence of red fluorescence, we predict that the following clones are likely expressing our synthetic proteins:

* SpyCatcher:CsgA (A) – 1, 2, 4, and 5
* CsgA:SpyCatcher (B) – 1, 6, and 7
* SpyTag:Laccase (C) – no fluorescent pellets observed, which was to be expected due to unsuccessful colony PCR
* Laccase:SpyTag (D) – 8, 11, and 14
* SpyTag:Cutinase (E) – 4 & 5
* Cutinase:SpyTag (F) – 1, 2, 3, 4, and 5

DNA was then isolated from all pelleted cells using the EZ-10 Spin Column Plasmid Purification Kit from BioBasic Inc. With isolated plasmid DNA, we then repeated the same diagnostic PCR protocol (**Figure 7**).

![Responsive image](https://static.igem.org/mediawiki/2019/3/3f/T--Western_Canada--results7.jpg)

**Figure 7. Gel electrophoresis of PCR products from isolated plasmid DNA from induced *E. coli* cultures.** 1 µL of the products from the diagnostic PCR of the plasmid insertion site (**Figure 4**) of each assembled plasmid was run on a 1.4-% agarose gel at 120 V for 40 minutes. PCR products yielding electrophoresis bands of the right size are identified with green circles.

These PCR results further confirm the fluorescence observations, with red pellets corresponding with a PCR amplicon band of the correct size for gene insertion and plasmid assembly. The exception to this is clone 1 of the SpyTag:Cutinase construct, which yielded a correctly-sized band without visible fluorescence in the pelleted cells. This discrepancy suggests that while the gene SpyTag:Cutinase gene was successfully inserted into the plasmid, there may have been spontaneous mutations to the pBAD promoter region or the mRFP cistron/RBS.

To further probe each assembled construct for successful assembly and overall integrity, we digested samples of each plasmid with the high-fidelity restriction endonuclease EcoRV-HF from New England Biolabs. For each plasmid, this restriction enzyme should yield a banding pattern that differs from that of pAGE 2.0 without any inserted fragment (**Figure 8**).

![Responsive image](https://static.igem.org/mediawiki/2019/7/74/T--Western_Canada--results9.jpg)

**Figure 8. Gel electrophoresis of EcoRV-HF restriction endonuclease digest products from isolated plasmid DNA from induced *E. coli* cultures.** Top panel: expected banding pattern from an EcoRV digest of each assembled plasmid. This figure was generated using Benchling’s virtual digest feature. Middle and bottom panels: 300 ng of each isolated plasmid was digested with 10 units of EcoRV-HF restriction enzyme in a 20-µL solution containing 2 µL of CutSmart. After incubating for one hour at 37°C followed by 20 minutes at 65°C, the entire solution was run on a 1.4-% agarose gel at 120 V for 40 minutes. Lanes with the expected banding pattern are identified with green circles.

The restriction digest results largely confirm the PCR and fluorescence results described above, however there are some unexpected banding patterns for some clones. Surprisingly, the banding patterns for clones 8 and 11 of the Laccase:SpyTag construct are closer to the pattern expected for SpyTag:Laccase. As with the PCR result for SpyTag:Cutinase clone 1, the banding pattern suggests a properly structured plasmid, which contrasts with the absence of fluorescence. These unusual findings require further assays, such as sequencing, to elucidate the underlying plasmid structure.

### *Sequencing of insertion regions*

The PCR reaction products for each fragment were submitted for Sanger sequencing at the Robarts Research Institute DNA Sequencing Facility using the same primers as for PCR. The sequence data we received was analyzed by mapping each read to the corresponding reference sequence and disagreements from the reference sequence were highlighted (**Figure 9**). The raw sequence results are available at the end of the lab [Notebook](https://2019.igem.org/Team%3AWestern_Canada/Notebook).

![Responsive image](https://static.igem.org/mediawiki/2019/b/ba/T--Western_Canada--ResultsA1.png)
![Responsive image](https://static.igem.org/mediawiki/2019/2/27/T--Western_Canada--ResultsA2.png)
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![Responsive image](https://static.igem.org/mediawiki/2019/4/46/T--Western_Canada--ResultsB1.png)
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![Responsive image](https://static.igem.org/mediawiki/2019/a/ad/T--Western_Canada--ResultsD8.png)
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![Responsive image](https://static.igem.org/mediawiki/2019/a/ae/T--Western_Canada--ResultsD14.png)
![Responsive image](https://static.igem.org/mediawiki/2019/4/40/T--Western_Canada--ResultsE1.png)
![Responsive image](https://static.igem.org/mediawiki/2019/0/04/T--Western_Canada--ResultsE4.png)
![Responsive image](https://static.igem.org/mediawiki/2019/f/fe/T--Western_Canada--ResultsE5.png)
![Responsive image](https://static.igem.org/mediawiki/2019/4/4c/T--Western_Canada--ResultsF1.png)
![Responsive image](https://static.igem.org/mediawiki/2019/5/5f/T--Western_Canada--ResultsF2.png)
![Responsive image](https://static.igem.org/mediawiki/2019/5/50/T--Western_Canada--ResultsF3.png)

**Figure 9. Consensus sequences from bidirectional Sanger sequencing of the insertion sites of each assembled plasmid.** Forward and reverse Sanger sequencing results of the PCR amplicon of the insertion site of each assembled plasmid were aligned to their corresponding reference sequences. The consensus of each pair of sequencing reads was annotated, regions which aligned with the reference were shaded in grey, and disagreements (including ambiguous base calls and no read coverage) were highlighted in black. Figure was generated using Geneious version 2019.2 by Biomatters (available from www.geneious.com).

This is how far our team has gotten with our iGEM project. Next year, Western Canada’s iGEM team will continue where we left of by transferring clones with correct sequences to expression strains and performing functional experiments to characterize each part.

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