iGEM 2020 — Team DeNovocastrians

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# Team:DeNovocastrians/Notebook

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Project

[Description](https://2020.igem.org/Team%3ADeNovocastrians/Description)
[Experiments](https://2020.igem.org/Team%3ADeNovocastrians/Experiments)
[Results](https://2020.igem.org/Team%3ADeNovocastrians/Results)
[Notebook](https://2020.igem.org/Team%3ADeNovocastrians/Notebook)
[Engineering](https://2020.igem.org/Team%3ADeNovocastrians/Engineering)
[Implementation](https://2020.igem.org/Team%3ADeNovocastrians/Implementation)
[Contribution](https://2020.igem.org/Team%3ADeNovocastrians/Contribution)

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[Safety](https://2020.igem.org/Team%3ADeNovocastrians/Safety)
[Human Practices](https://2020.igem.org/Team%3ADeNovocastrians/Human_Practices)
[Education](https://2020.igem.org/Team%3ADeNovocastrians/Education)
[Poster](https://2020.igem.org/Team%3ADeNovocastrians/Poster)
[Awards](XXXXX)

**Laboratory Notebook**

**Week 1 (03/08/20-07/08/20)**

**Aakash**: Media preparations. Overnight cultures of Rhodococcus strain 9, Rhodococcus strain 33, E. coli and ADP1. Growth curves in LB media/M9+(15g/L)Benzoate media. E.coli::PTTQ18/PETDUET/PACYC plasmid extractions.

**Ruby**: Growth curves of Rhod 9, Rhod 33 and ADP1 in LB and M9 media. Primer design. Prepared PACYC Duet strain DH5α, PetDuet strain DH5α and PTTQ18 strain DH5α for future plasmid preparations. Streaked PACYC and PetDuet onto 30ug/ml CAM agar plates. Streaked PTTQ18 onto 100ug/ml AMP agar plate. Prepared overnight cultures in M9 media - pipette inoculated 100ug/ml Amp M9 solution with PTTQ18, pipette inoculated 50ug/ml CAM M9 solution with PACYC and PetDuet.

**Darcy&Cate**: Strain 33&9 were cultured for 2days to allow for genomic DNA extraction.Extraction of Rhod 9 and Rhod 33 genomic DNA by using the DNeasy Ultraclean microbial kit. PCR was undertaken on the extracted gDNA, strain9&33 gDNA was diluted down to 10ng/uL Rhod Strain 9
g

**Week 2 (10/08/20-14/08/20)**

**Aakash**: B.subtilis(168), E.coli(BW25113), Synechocystis overnight cultures. B.subtilis(168) and E.coli(BW25113) growth curves in 10mM/100mM Benzoate.

**Ruby**: PCR of Rhod 9 benE, his tagged Rhod 9 benE, ADP1 benE and his tagged ADP1 benE.

**Darcy&Cate**: A PCR was undertaken on the BenABCD cluster of Strain 9 and E.ferg. This was was repeated twice. And gel electrophoresis was undertaken on all PCR samples

**Week 3 (17/08/20-21/08/20)**

**Aakash**: Synechocystis PCC 6803 Growth Curve in 10mM/100mM Benzoate.

**Ruby**: PCR Rhod 9 benE, his tagged Rhod 9 benE, E. Ferg benE. Checked E.Ferg g-block. Ethanol precipitation of Rhod 9 benE and his tagged Rhod 9 benE PCR products.

**Darcy&Cate**: This PCR was a repeat of the previous week (12/08/2020) with 1 more cycle of PCR. Another PCR was run with lower annealing temp and high GC buffer. Gel were run for all PCR samples.

**Week 4 (24/08/20-28/08/20)**

**Aakash**: E.coli BL21, E.coli BW25113, B.subtilis, Acinetobacter ADP1, Rhod strain 9, Rhod strain 33 overnight cultures. Growth curves of E. coli BL21, E. coli BW25113, B. subtilis, Acinetobacter baylyi ADP1 in 10mM/100mM/1M Benzoate (Microtiter plate).

**Ruby**: PCR Cleanup of ADP1 benE and ADP1 benE his PCR products.

**Darcy&Cate**: PCR clean up was done for the PCR products of ADP1,E.ferg and Rhod St.9. This was follwed by a digestion and ligation reation to clone the PCR products into the PTTQ18 plasmid.

**Week 5 (31/08/20-4/09/20)**

**Aakash**: Rhod strain 9/Rhod strain 33/E.coli/ADP1/168 overnight cultures. Catechol media prep. Synechocystis subculture. Growth curves of E. coli BL21, E. coli BW25113, B. subtilis, Acinetobacter baylyi ADP1 in 10mM/100mM/1M Benzoate (Microtiter plate). E. coli (BL21)::PTQ118 plasmids extraction.

**Ruby**: Digests - ADP1 benE and ADP1 his benE into pTTQ plasmid, Rhod 9, Rhod 9 his and E. Ferg benE into pET30a plasmid. Ligation. Transformations of plasmids into competent E. coli cells. Transformed E. coli cells grown on selection plates.

**Darcy&Cate** The ligation reaction was repeated with antartic phosphatase. A transfromation was attempted but failed. After this, a digestion was done on the restriction enzymes to check function.

**Week 6 (07/09/20-11/09/20)**

**Aakash**: E. coli BL21, E. coli BW25113, B. subtilis, Acinetobacter baylyi ADP1 overnight cultures and growth curves in serial diluted 1M Catechol (microtitre plate).

**Ruby**: PCR confirmation of transformations. Overnight cultures of transformed cells. Extracted plasmids from overnight cultures. Ran digests to check for benE in plasmids.
Darcy&Cate: A digestion reaction was repeated with new enzymes, following this a overnight ligation reaction was undertaken.

**Week 7 (14/09/20-18/09/20)**

**Aakash**: Rhodococcus 9, Rhodococcus 33 and ADP1 overnight cultures and growth curves in serial diluted 1.5M Catechol (microtitre plate).

**Ruby**: PCR of ADP1 benE upstream, benE downstream, benK downstream and benK upstream fragments for knockout PCR. PCR cleanup of products. Overnight cultures of plasmid lab stock cultures: KH142, IG1110 and IG245. Plasmid preps on these overnight cultures.

**Darcy&Cate**: The cloned plasmid was sent off for sequencing and bioinfomatics of the benABCD cluster was undertaken.

**Luke**: using ADP1 gDNA(isolated from Ruby “Igem member”), made 3 PCR to isolate fragments from gDNA 0.6ul of gDNA in each reaction, with extension time 70°C for 25 seconds. To isolate CatM binding region, CatM promoter and BenM binding region.

Run on ethdium Bromide gel and CatM promoter and BenM binding region, showed bands at correct length. CatM binding region showed no DNA present in lane.

BenM, GFP, mCherry and CatM gene sequences arrived from Twist.

Created working solutions of all fragments to contain 1ng/ul for all regions, which was used for PCR, to join all fragments together in one reaction.
Run on ethidium bromide gel and contained no bands at desired length
Performed overlap extension PCR, by using 1ul of 1ng/ul concentration for each fragment in PCR. 6 fragments with corresponding primers, to contain overlap regions from primers.

| Reaction | Template | Primer 1 | Primer 2 | Concentration ng/uL |
| --- | --- | --- | --- | --- |
| 1 | mcherry gene | 1FWD | 1REV | 197 |
| 2 | catm binding region | 2 REV | geneless REV | 23 |
 3 | catm binding region | 2 REV | geneless REV | 23 || 4 | catm gene | 4FWD | 4REV | 191 |
| 5 | benm gene | 5FWD | 5REV | 42.2 |
| 6 | benm promoter/binding region | 6REV | GENELESS FWD | 60 |
| 7 | GFP gene | 7FWD | 7REV | 44.6 |

Run on ethidium bromide gel

![](https://static.igem.org/mediawiki/2020/f/f8/T--DeNovocastrians--notebook_picture3.png)

Run on ethidium bromide gel,

![]()

each band was at the correct location, although there was other bands and smear present within gel

**Week 8 (21/09/20-25/09/20)**

**Aakash**: Rhodococcus 9, Rhodococcus 33 and ADP1 overnight cultures and growth curves in serial diluted 1M Benzoate (microtitre plate).

**Ruby**: PCR on IG245 plasmid amplifying FRT sites for gene knockouts. PCR cleanup of products obtained.

**Darcy&Cate**: ADP1 and Rhod9 DNA was amplifed with new primers, this was then used in a digestion and ligation reaction.

**Luke**;Performed overlap extension PCR, by using 1ul of 1ng/ul concentration for each fragment in PCR.

Trying to join mCherry to Catm binding(geneless), mCherry to Catm binding(whole construct), Gfp to BenM promoter(geneless and whole construct), CatM promoter to CatM gene, all fragments for whole construct, all fragments for geneless construct.

Run on 45ul on thyazoile orange gel and all lanes contained nothing(not enough DNA used).
Used remaining 5ul for amplification with corresponding primers for each potentially joined fragments.

Performed gel extractation for GFP-BenM promoter(geneless), GFP-BenM promoter and CatM promote-CatM gene. All other overlap extension PCR did not contain band in the correct position.

Solutions below followed optimization protocol for overlap extension PCR(Hilgarth and Lanigan, 2020).

Trying to join mCherry to Catm binding(geneless), mCherry to Catm binding(whole construct), Gfp to BenM promoter(geneless and whole construct), CatM promoter to CatM gene, all fragments for whole construct, all fragments for geneless construct.

run on tyhazoile orange gel

mCherry-CatM binding (geneless), failed(lane 2)

m-Cherry-CatM binding(whole construct), success, although large amount of smearing and other bands(lane 3)

whole construct, not in correct position, not at correct position, although large amount of smearing and other bands(lane 4)

geneless construct, not in correct position, not at correct position, although large amount of smearing and other bands(lane 5)

from whole construct and geneless construct, removed 5ul from reraction and amplified using primers for whole fragment(hopefully to purify, less smearing, less bands). then run reaction on thiazoile orange.

![]()

Same result, large amount of smearing and other bands present.
Gel extracted geneless construct band(lane 2), whole fragment at fully constructed length 4500(although there was no band) and at bright band 3500(lane 3).
Amplified via PCR using just 0.5ul from each solution, then run on gel, no DNA present within lane.

Using potential extracted geneless solution, performed restriction, ligation into pUC19 plasmid and transformed into DH5alpha cells.
Was unsuccessful, no transformations.

Performed overlap extension PCR following optimized protocol(Hilgarth and Lanigan, 2020). Of CatM promoter with CatM binding and BenM promoter with GFP.

**Week 9 (28/09/20-02/10/20)**

**Aakash**: Rhodococcus 9, Rhodococcus 33 and ADP1 overnight cultures and growth curves in serial diluted 250mM Benzoate (microtitre plate). E. coli BW25113 benzoate/catechol MIC's. benE plasmid preps.

**Ruby**: Plasmid preparation of overnight cultures of transformed cells set up by supervisor Catherine. Digests to confirm the presence of target genes within the plasmids.

**Darcy&Cate**:OVernight cultures were preared which were then used for a colony PCR. The colony PCR was undertaken to identify sucesfully cloned plasmids. After unsucesfully identify any transformats, a digest and ligation experiment was repeated on benABCD from Rho9 and ADP1.

**Luke**; Performed overlap extension PCR following optimized protocol(Hilgarth and Lanigan, 2020). Of mCherry with CatM binding and BenM promoter with BenM.

Run on thyazoile orange gel.

![](https://static.igem.org/mediawiki/2020/d/dd/T--DeNovocastrians--notebook_picture5.png)

Gel extracted mCherry with CatM binding(lane 2), BenM promoter with BenM(lane 3), and BenM promoter with GFP(lane 5).

Performed overlap extension PCR with 3500 whole construct and BenM promoter-GFP.

Run amplification of 3500 whole construct and BenM promoter-GFP, BenM-BenM binding, mCherry-CatM binding, mCherry-CatM binding(geneless) and GFP-BenM binding.photo

Run all amplifications on ethidium bromide gel,

![](https://static.igem.org/mediawiki/2020/5/50/T--DeNovocastrians--notebook_picture6.png)

BenM-BenM binding and mCherry-CatM binding had bands in correct positions(lane 2 and 3), although contained smear and other bands. All other overlap extension PCR failed, thus having light bands in incorrect positions.

Amplified CatM binding region(geneless) and BenM binding(geneless) from ADP1.

Performed overlap extension PCR of BenM-BenM binding with GFP and mCherry-CatM binding with CatM promoter.

Run on ethidium bromide gel BenM-BenM binding with GFP(lane 2), mCherry-CatM binding with CatM promoter(lane 3), CatM binding region(geneless)(lane 4) and BenM binding(geneless) from ADP1(lane 5), CatM binding region(geneless) with mCherry(lane 7) and BenM binding(geneless) with GFP(lane 8).

![](https://static.igem.org/mediawiki/2020/f/ff/T--DeNovocastrians--notebook_picture7.png)

BenM-BenM binding with GFP, showed light band of correct size, although other bands were present.(lane 3)
mCherry-CatM binding with CatM promoter, contained band close to correct size, although contained large amount of smearing and other bands.(lane 4)
CatM binding region(geneless) and BenM binding(geneless) from ADP1, were unsuccessful with no DNA in lane 5 and 6.
mCherry and BenM binding(geneless) with GFP, were unsuccessful with no band in correct position.(lane 7 and 8)

Checked primer annealing temperature to observe if reason for other bands and smears within gels.
Created gradient PCR with all 9 separate fragments, 72 in total from 62°C
to 50°C.
Run on gel for 1 hour at 100 volts, with 2 sets of lanes on gel. All solutions run off gel and hit wells, thus unusable.

2nd try;
Created gradient PCR with all 9 separate fragments, 72 in total from 62°C
to 50°C.
run on gel at 100 volts for 20 minutes.

 photo
Primer annealing for all primer much higher than expected

![](https://static.igem.org/mediawiki/2020/5/52/T--DeNovocastrians--notebook--gradient_PCR.tiff)

Each fragment is split between DNA ladder. Top left,section 1=fragment 1 from 60°C-50°C, then top middle, section2=fragment 2 from 60°C-50°C, then top right section 3=fragment 3 from 60°C-50°C, then bottom right section 4=fragment 4 from 60°C-50°C, then section 5 and 6.

![](https://static.igem.org/mediawiki/2020/b/b0/T--DeNovocastrians--notebook--gradient_PCRsmall.tiff)

Small gel. Top left section= section 7=fragment 7 from 72°C -60°C, then bottom right, section 8=fragment geneless BenM binding from 72°C -60°C, then right section starting from top then going to 2 on bottom right, section 9=fragment geneless catM binding from 72°C -60°C.

all lanes contained bands, except for fragment 7 which contained bands in 5 lowest temperatures(temp up to 66.1°C)

Performed a gradient overlap extension PCR for BenM-BenM binding with GFP and mCherry-CatM binding with CatM promoter. At temperatures 65, 63.5 58.9 and 57°C.
And amplified potential geneless fragment at temperatures 65, 63.5 58.9 and 57°C.

![](https://static.igem.org/mediawiki/2020/d/d8/T--DeNovocastrians--notebook_picture8.png)

Band at the correct position for BenM-BenM binding-GFP, although other bands and large amount of smearing(lane 2-5).
No band but smearing across regions for mCherry-catM binding-CatM promoter and geneless construct(lane6-9).

**Week 10 (05/10/20-09/10/20)**

**Ruby**: Digests of ADP1 transformations (cultures hadn't grown well enough to have plasmid preparations undertaken in previous week - Aakash undertook plasmid preparations on these cells. PCR reactions to stich together upstream, benE/benK and downstream gene fragments for gene knockouts.

**Darcy&Cate**: A colony PCR was undertaken on the transformants prepared in the previous week.

**Luke**;run ladder, BenM-BenM binding+GFP amplified fragment at 65, CatM, CatM binding+ Catm Promoter, small G(geneless construct), 40ul of each

Bands were not present in correct locations.

Aimed to furthy purify BenM-BenM binding and mCherry-CatM binding.
Placed in 2% agarose gel with thyazoile orange
running at 50 volts, 30 minutes, checked.
50 volts, 1 hour, checked.
70 volts, 30 minutes, checked.
30 minutes.
Then performed gel extraction

Then performed overlap extension PCR with BenM-BenM binding with GFP and mCherry-CatM binding with CatM promoter.

run 45ul of yesterdays reactions;
BenM-BenM binding with GFP and
mCherry-CatM binding with CatM promoter,
on thyazoile oranage gel.
100 volts for 1 hour.
Nothing within lanes, bp ladder present.

run PCR master mix of 5ul of reaction left over, amplyfiying sections
run on thyazoile orange gel.
Nothing within lanes, ladder present.

**Week 11 (12/10/20-16/10/20)**

**Aakash**: E.coli knockout strains with inserted benE/benABCD clusters overnight cultures and growth curves in serial diluted 125mM Benzoate (microtitre plate). E. coli BW25113 (benE knockout strain) benzoate/catechol MIC's.

**Ruby**: Made calcium chloride competent E. coli BW 25113 cells (BenE knockout equivalent cells). Set up overnight cultures of WT E. coli 25113 on no selection media, Knockout E. coli cells in Kan and E. coli transformed with pTTQ18 plasmid in Amp. Performed transformations inserting plasmids containing Rhod 9 BenE, E. Ferg BenE, ADP1 benABCD and Rhod 9 benABCD into the competent cells.

**Luke**: from amplified mCherry-CatM binding performed restriction ligation using Ecor1 site and smar1 for blunt end, into PWH1266 plasmid. Transformed using ligation reactions, colonys present in control with no ligation reaction in them. tryed colony PCR and plasmid prep with restrictation digest. No plasmid with dedsired insert length was found.

obtained new primers that do not contain overlap regions.

Using 1ul solution containing CatM promoter-CatM gene and 1ul solution containing BenM binding-BenM gene(which contained many bands and large amount of smearing). Amplified these fragments using new primers and annealing temperature at 58°C, this resulted in less smearing and bands present within gel lane.

![](https://static.igem.org/mediawiki/2020/2/26/T--DeNovocastrians--notebook_picture9.png)

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# Team:DeNovocastrians/Engineering

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Project

[Description](https://2020.igem.org/Team%3ADeNovocastrians/Description)
[Experiments](https://2020.igem.org/Team%3ADeNovocastrians/Experiments)
[Results](https://2020.igem.org/Team%3ADeNovocastrians/Results)
[Notebook](https://2020.igem.org/Team%3ADeNovocastrians/Notebook)
[Engineering](https://2020.igem.org/Team%3ADeNovocastrians/Engineering)
[Implementation](https://2020.igem.org/Team%3ADeNovocastrians/Implementation)
[Contribution](https://2020.igem.org/Team%3ADeNovocastrians/Contribution)

[Parts](https://2020.igem.org/Team%3ADeNovocastrians/Parts)
[Safety](https://2020.igem.org/Team%3ADeNovocastrians/Safety)
[Human Practices](https://2020.igem.org/Team%3ADeNovocastrians/Human_Practices)
[Education](https://2020.igem.org/Team%3ADeNovocastrians/Education)
[Poster](https://2020.igem.org/Team%3ADeNovocastrians/Poster)
[Awards](XXXXX)

**Engineering Success**

The *benE* gene is known to encode a benzene-H+ symporter protein (Choudary et al., 2017). This gene was a target for our project as it enables bacteria to import benzene/benzoate from their environments to use as a carbon source in their energy generation. We extracted *benE* genes from *Rhodococcus strain 9, Acinetobacter baylyi (ADP1)* and *Escherichia fergusonii*. The genes were successfully ligated into a pTTQ18 plasmid. Of these plasmids, those containing the *benE* gene from *E. fergusonii* and *Rhodococcus strain 9* were successfully transformed into an *E. coli benE* knockout strain.

![](https://static.igem.org/mediawiki/2020/c/cf/T--DeNovocastrians--LB_growth_curve.png) Figure 1: *E. coli* strains growth in LB + benzoate.

Growth curve in LB + benzoate: The strains transformed with *benE* were able to grow in a concentration of 15.625mM benzoate. The *benE* knockout strain and the *benE* knockout strains transformed with the plasmid only, were unable to grow at the 15.625mM benzoate concentration. This suggested that the *benE* transporter enables cells to import and utilise benzoate as a carbon source.
![](https://static.igem.org/mediawiki/2020/d/de/T--DeNovocastrians--HPLCbenzo.png) Figure 2: *E. coli* strains degradation of benzoate.

HPLC Curve: This result demonstrated that the cells transformed with the *benE* genes were able to successfully transcribe and translate the transporter, this enabled them to import benzoate from their extra-cellular environment. Compared to the *benE* knockout cells, the strains containing *benE* genes (from both *Rhodococcus strain 9* and *E. fergusonii*) had utilised benzoate from their environment as a carbon source, shown here by the reduction in benzoate readings in their associated curves compared to the baseline 15mM benzoate curve. The cells transformed with *benE* from *E. fergusonii* imported the highest levels of benzoate compared to the *benE* knockout cells.

Taken together these results confirm that *benE* acts as a chemical trans-membrane transporter that enables cells to import benzene/benzoate from their environments. Further, the results suggested that *benE* may enable cells to survive in higher concentrations of benzene/benzoate, by enabling them to import and use the chemical as a carbon source for energy generation. Therefore, we were able to demonstrate engineering success for the parts [BBa\_K3694028](http://parts.igem.org/Part%3ABBa_K3694028) (*benE E. fergusonii*) and [BBa\_K3694029](http://parts.igem.org/Part%3ABBa_K3694029) (*benE Rhod. 9*).

### References

**Choudary, A., Purohit, H. and Phale, P. S.** (2017). Benzoate transport in Pseudomonas putida CSV86. *FEMS Microbiology Letters* **364(12)**, 1-9.

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# Team:DeNovocastrians/Experiments

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Team

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[Attributions](https://2020.igem.org/Team%3ADeNovocastrians/Attributions)
[Collaborations](https://2020.igem.org/Team%3ADeNovocastrians/Collaborations)

Project

[Description](https://2020.igem.org/Team%3ADeNovocastrians/Description)
[Experiments](https://2020.igem.org/Team%3ADeNovocastrians/Experiments)
[Results](https://2020.igem.org/Team%3ADeNovocastrians/Results)
[Notebook](https://2020.igem.org/Team%3ADeNovocastrians/Notebook)
[Engineering](https://2020.igem.org/Team%3ADeNovocastrians/Engineering)
[Implementation](https://2020.igem.org/Team%3ADeNovocastrians/Implementation)
[Contribution](https://2020.igem.org/Team%3ADeNovocastrians/Contribution)

[Parts](https://2020.igem.org/Team%3ADeNovocastrians/Parts)
[Safety](https://2020.igem.org/Team%3ADeNovocastrians/Safety)
[Human Practices](https://2020.igem.org/Team%3ADeNovocastrians/Human_Practices)
[Education](https://2020.igem.org/Team%3ADeNovocastrians/Education)
[Poster](https://2020.igem.org/Team%3ADeNovocastrians/Poster)
[Awards](XXXXX)

**Experiments**

**Benzene Biosensor**

### Construction of biosensor G-Block

![](https://static.igem.org/mediawiki/2020/e/eb/T--denovocastrians--bencatsen_whole_construct.png)- Figure 1 whole construct of benzene and catechol biosensor.
![](https://static.igem.org/mediawiki/2020/8/85/T--denovocastrians--geneless_construct.png)- Figure 2 gene less construct of benzene and catechol biosensor, control.

To construct absolute construct shown in figure 1 and gene less construct shown in figure 2. Genes GFP, mCherry, CatM and BenM coding sequences were ordered from twist. The coding regions CatM binding region, CatM promoter, BenM promoter/binding region and gene less counterparts were amplified out of ADP1 genome to contain overlap regions able to join catM binding to BenM binding.

Merck Hot StarTaq DNA polymerase kit with ADP1 genome and 10um of primers for each region were amplified with annealing temperature at 53°C. PCR clean up was done via QIAGEN PCR clean up DNaeasy, ultraclean, microbial kit.

Overlap extension PCR was done following optimization protocol (Hilgarth and Lanigan, 2020) with annealing temperature of 53°C for overlap stage and 57°C for amplification. Gel extraction was done via QIAGEN, QIAquick, in thyazoile orange gel, viewed under UV light. Each fragment was joined/constructed individually, to make constructs in figure 1 and 2, ie mCherry joined to catM binding region, then construct joined to CatM promoter, etc.

Restriction and ligation utilised HindIII and EcorI sites placed from primers to restrict and ligate into plasmid vector pUC19 , with 100um of vector and 300um of insert.

Transform by using thawed DH5α and 10-20ul of ligation reaction, incubate for 10 minutes on ice, heat pulse for 2 minutes, add 150ul of LB media, incubate at 37°C for 1 hour, plate on selective media.

Transform by using thawed DH5α and 10-20ul of ligation reaction, incubate for 10 minutes on ice, heat pulse for 2 minutes, add 150ul of LB media, incubate at 37°C for 1 hour, plate on selective media.

**Cloning *benE***

### DNA Extraction

DNA of *E. fergusonii* was synthesized in a laboratory.
DNA of *Rhodococcus strain 9* and *Acinetobacter baylyi ADP1* was extracted using the DNeasy ultraclean microbial kit (suggested protocol followed).

### Gene Amplification

***Rhodococcus strain 9* and *Acinetobacter baylyi ADP1***

PCR Mix: 5ul - 10x buffer for KOD + 3ul - MgSO4 (25 mM) + 5ul - dNTPs (2 mM each) + 31ul - mQ water + 1.5ul - 5' primer (10 mM) + 1.5ul - 3' primer (10mM) + 2ul - gDNA (40 ng).

Using *Rhodococcus strain 9* and *Acinetobacter baylyi ADP1* gDNA
Primers=
*Rhodococcus strain 9*:
FWD: GTACGAATTCCatgaacccatccaccac
RVS: CTAGAAGCTTtcacgacacctggacg
*Acinetobacter baylyi ADP1*:
FWD: AGTCGAATTCCatgacaaccctgttaaaaac
RVS: CGTCGGATCCttagctggctttgggtt

**PCR run:**
95ºC - 2 min
95ºC - 20 sec
54ºC - 10 sec
70ºC - 30 sec
Repeat steps 2-4 for 30 cycles
70ºC - 5 mins
hold at 12ºC

**Digests**
Digests were performed on *benE* from *E. Fergusonii, Rhodococcus strain 9, Rhodococcus strain 9-his, Acinetobacter baylyi ADP1* and *Acinetobacter baylyi ADP1-his.*

![](https://static.igem.org/mediawiki/2020/b/bc/T--Denovocastrians--Digest_1_image.jpg)

Water bath samples at 37°C for 1 hour.
Added 5ul rSAP to plasmid to prevent re-ligation.
PCR cleanup was performed on samples using the QIAquick PCR and Gel Cleanup Kit.

**Ligation**
Ligation reactions:

![](https://static.igem.org/mediawiki/2020/b/b3/T--Denovocastrians--ligation_graph.jpg)

Incubate at 16°C overnight.

**Transformations**
50ul CaCl2 competent cells + 5ul plasmid DNA (pTTQ18 control, pTTQ18::*Acinetobacter baylyi ADP1 benE*, pTTQ18::*Acinetobacter baylyi ADP1-his benE*, pTTQ18::*Rhodococcus strain 9 benE*, pTTQ18::*Rhodococcus strain 9-his benE* and pTTQ18::*E. Fergusonii benE*)
Incubate on ice 15 mins.
Heat shock at 42°C for 45 seconds.
Add 1ml LB to each tube.
Shake in 37°C room for 5 mins.
Spin at 1700g for 1 min.
Pour off most supernatant (leave around 100ul).
Resuspend in the remaining supernatant
Plate onto agar plates containing selective media (100ul/ml Amp)

**Overnight cultures**
Overnight cultures were set up for each of the transformation samples in 100ug/ml Amp M9 media. Samples were left overnight on shaking platform in 37°C room.

**Plasmid Extraction**
Plasmids were extracted from the overnight cultures of the transformations using the QIAprep Spin Miniprep Kit (suggested protocol was followed).

![](https://static.igem.org/mediawiki/2020/2/29/T--Denovocastrians--digest_confirmations.jpg)

**Microbial growth and Minimum Inhibitory Concentration (MIC) assays**

### **Working safely**

* Ensure all PPE (gloves, lab coat, goggles, enclosed shoes, hair tied back) is worn.
* Ensure work area is wiped down with 70% ethanol.
* Ensure all materials are prepared, collected and have been autoclaved.
* Ensure all utensils are within reach.
* Label all materials and cultures with name of contained, date, experimenter name, extra information.
* Work close to the Bunsen burner (using blue flame) in the convection current zone. Use the blue flame setting to flame metal loops, neck of glass bottles/containers/tubes 2-3 times.
* Safely handle and dispose of hazardous materials in the correct manner according to Material Safety Data Sheet (MSDS).
* Wipe down bench with 70% ethanol when finished with experiments.
* Discard of bacteria-containing media and contaminated materials appropriately (e.g. soak in bleach, autoclave).

### **Media**

**Luria Broth (LB) (800ml) (Negative and positive control media)**1. LB media: 4g NaCl + 4g Yeast extract + 8g Tryptone + 800ml Milli-Q water.
2. Stir well and auto clave.

**(5X) M9 salts minimal media (100ml)**1. 5.25g M9 salts (by AMRESCO) + 100ml Milli-Q water.
2. Stir well and auto clave.

**(15g/L) Benzoate (10ml)**1. 0.15g sodium benzoate salts + 10ml Milli-Q water.
2. Filter solution through 0.2µm filter syringe.

**(2M) Benzoate** 1. 2.882g sodium benzoate salts + 10ml Milli-q water.
2. Filter solution through 0.2µm filter syringe.

**(2M) Catechol**1. 2.202g catechol salt + 10ml Milli-q water.
2. Filter solution through 0.2µm filter syringe.

**(3M) Catechol**1. 990mg catechol salts + 3ml Milli-Q water.
2. Filter sterilise with 0.2µm filter syringe.

**M9 minimal salts + (15g/L) Benzoate (500ml) growth curves**1. 100ml stock (5X) M9 salts media + 5ml sodium benzoate (15g/L concentration) + 1ml (1M) MgSO4 + 500ul (20mM) CaCl2 + 393.5ml Milli-q water.

**10mM/100mM/1M Benzoate growth curves media (100ml total volumes):**1. M9 + glucose: 20µl M9 (5X) stock solution + 2ml (20%) glucose + 200µl (1M) MgSO4 + 100µl (0.1M) CaCl2 + 97.68ml Milli-q water.
2. M9 + (10mM) sodium benzoate: 20ml M9 (5X) stock solution + 0.5ml (2M) benzoate + 200µl (1M) MgSO4 + 100µl (0.1M) CaCl2 + 99.18ml Milli-q water.
3. M9 + (100mM) sodium benzoate: 20ml M9 (5X) stock solution + 5ml (2M) benzoate + 200µl (1M) MgSO4 + 100ul (0.1M) CaCl2 + 94.68ml Milli-q water.
4. M9 + (1M) sodium benzoate: 20ml M9 (5X) stock solution + 50ml (2M) benzoate + 200µl (1M) MgSO4 + 100ul (0.1M) CaCl2 + 49.68ml Milli-q water.

**10mM/100mM/1M Catechol growth curves media (20ml total volumes):**1. M9 + glucose (50ml): 10ml M9 (5X) stock solution + 1ml (20%) glucose + 100µl (1M) MgSO4 + 50µl (0.1M) CaCl2 + 48.84ml Milli-q water.
2. M9 + (10mM) catechol: 4ml M9 (5X) stock solution +100ul (2M) catechol + 40µl (1M) MgSO4 + 20µl (0.1M) CaCl2 + 15.84ml Milli-q water.
3. M9 + (100mM) catechol: 4ml M9 (5X) stock solution + 1ml (2M) catechol + 40µl (1M) MgSO4 + 20µl (0.1M) CaCl2 + 14.94ml Milli-q water.
4. M9 + (1M) catechol: 4ml M9 (5X) stock solution + 10ml (2M) catechol + 40µl (1M) MgSO4 + 20µl (0.1M) CaCl2 + 5.94ml Milli-q water.

**Negative/Positive control media for Rhodococcus strains 9/33 & ADP1**1. M9 (1X) (100ml total) =20ml M9 (5X) + 200µl (1M) MgSO4 + 100µl (0.1M) CaCl2 + 79.7ml H2O.
2. M9 + 0.5% succinate (1.5ml total) =75µl (10%) succinate + 1.425ml M9 (1X).
3. M9 + 0.5% glucose (1.5ml total) =15µl (50%) glucose + 1.485ml M9 (1X).

### **Overnight cultures**

***Rhodococcus strains 9*** 1. Place 5ml of LB media into two 10ml falcon tubes.
2. Aseptically inoculate two nutrient agar plates with Rhodococcus strains 9 from -80°C frozen stocks.
3. Aseptically inoculate the two falcon tubes with Rhodococcus strain 9 picked from single colonies on plates or from frozen stocks.
4. Incubate cultures in 28°C shaker at 200-250RPM overnight.

***Rhodococcus strains 33*** 1. Place 5ml of LB media into two 10ml falcon tubes.
2. Aseptically inoculate two nutrient agar plates with Rhodococcus strains 33 from -80°C frozen stocks.
3. Aseptically inoculate the two falcon tubes with Rhodococcus strain 33 picked from single colonies on plates or from frozen stocks.
4. Incubate cultures in 28°C shaker at 200-250RPM overnight.

***Acinetobacter baylyi ADP1***1. Fill one 10ml falcon tube with 5ml of Luria Broth (LB).
2. Aseptically collect a single colony from ADP1 nutrient agar stock plate.
3. Inoculate into 10ml falcon tube containing 5ml LB.
4. Leave overnight in 37°C room on shaker.

***Escherichia coli (BW25113)*** 1. Aseptically collect a small amount of E. coli (BW25113) from -80°C frozen stocks.
2. Inoculate into 10ml falcon tube containing 5ml LB.
3. Leave overnight in 37'C room on shaker.

***Escherichia coli (BL21)*** 1. Aseptically collect a small amount of E. coli (Bl21) from -80°C frozen stocks or a single colony from nutrient agar stock plates.
2. Inoculate into 10ml falcon tube containing 5ml LB.
3. Leave overnight in 37'C room on shaker.

***Bacillus subtilis (168)*** 1. Fill one 10ml falcon tube with 5ml of Luria Broth (LB).
2. Aseptically collect a single colony of B. subtilis (168) from stock nutrient agar plate.
3. Inoculate into 10ml falcon tube containing 5ml LB.
4. Leave overnight in 37°C room on shaker.

***Synechocystis (PCC 6803)***1. Working in a biosafety cabinet.
2. Inoculate 50ml BG-11 media in a 250ml Erlenmeyer flask with 1ml Synechocystis pre-made stock culture.
3. Leave for 6 days in 24°C room on shaker.

### **Microbial growth assays**

**Growth curves of Rhodococcus strain 9, Rhodococcus strain 33, E. coli and Acinetobacter baylyi ADP1 in (150mg/L) Benzoate** 1. Added 50ml LB media to eight 125ml flasks.
2. Added 50ml M9+ benzoate media to eight 125ml flasks.
3. Inoculated 2 flasks of LB and 2 flasks of M9+ benzoate with 50µl of Rhodococcus strain 9, Rhodococcus strain 33, E. coli and ADP1 (1:1000 dilution).
4. Rhodococcus strains 9 and 33 were placed in a 24'C room on a 100RPM shaker.
5. E. coli and ADP1 were placed in a 37'C room on a 200RPM shaker.
6. Absorbances at 600nm were measured hourly for each inoculated growth flask using a spectrophotometer (LB media and M9+Sodium Benzoate media used as blanks).

**Growth curves of B. subtilis (168) and E. coli (BW25113) in 10mM/100mM Benzoate** 1. Added 50ml M9+glucose media to two 250ml flasks.
2. Added 50ml M9+ (10mM) benzoate media to four 250ml flasks.
3. Added 50ml M9+ (100mM) benzoate media to four 250ml flasks.
4. Inoculated 2 flasks of M9+ (10mM) sodium benzoate media and 2 flasks of M9+ (100mM) sodium benzoate media each with 500µl of B. subtilis (168) and E. coli (BW25113) (1:100 dilution).
5. B. subtilis (168) and E. coli (BW25113) flasks were placed in 37'C room on a 200RPM shaker.
6. Absorbances at 600nm were measured hourly for each inoculated growth flask using a spectrophotometer (M9+Sodium Benzoate media used as blank).

**Growth curves of Synechocystis (PCC 6803) in 10mM/100mM Benzoate** 1. In two flasks place 50ml BG-11 only media.
2. In two flasks place 50ml BG-11 + 10mM Benzoate.
3. In two flasks place 50ml BG-11 + 100mM Benzoate.
4. Inoculate each flask with 1:100 dilution of Synechocystis (4.5ml).
5. Take measurements at OD730 twice per day.
6. Monitor growth for 2-3weeks.

**Growth curves of E. coli BL21, E. coli BW25113, B. subtilis, Acinetobacter baylyi ADP1 in 10mM/100mM/1M Catechol (Microtiter plate)** 1. Place 100µl of M9+glucose into A1-10, B1-10, C1-10, D1-10.
2. Place 100µl of 2M Catechol into A3-4, B3-4, C3-4, D3-4.
3. Serial dilute 100ul from A/B/C/D-3/4 into A/B/C/D-5-10.
4. Place 100µl of 10mM M9 + Catechol into E1-2 and F1-2, E7-8 and F7-8.
5. Place 100µl of 100mM M9 + Catechol into E3-4 and F3-4, E9-10 and F9-10.
6. Place 100µl of 1M M9 + Catechol into E5-6 and F5-6, E11-12 and F11-12.
7. Inoculate 1µl of E. coli BL21 into row A, E. coli BW25113 into row B, B. subtilis 168 into row C and Acinetobacter baylyi ADP1 into row D into respective wells.
8. Monitor absorbance at 37'C for 41hrs in plate reader.

**Growth curves of Rhodococcus strain 9, Rhodococcus strain 33, E. coli and Acinetobacter baylyi ADP1 in Benzoate (Microtiter plate)** 1. Place 50µL of M9(1X) minimal media into columns 2-10.
2. Place100µl positive control media (M9 + 0.5% succinate, M9 + 0.5% glucose) into column 12.
3. Place 100µl 3M Benzoate into column 1, then serial dilute up to column 10.
4. Add negative control media (M9 + 0.5% succinate, M9 + 0.5% glucose) to column 11.
5. Add 50µl cells to columns 1-10 and column 12.
6. Monitor absorbance at 37'C for 41hrs in plate reader.

**E. coli BW25113 knockout strains (+ inserted benABCD/E) growth curves in 125/62.5/31.25/15.625mM benzoate (microtitre plate)**1. Strains= 1. E.coli BW25113 (WT), 2. E.coli BW25113 (Δydco) keoi:75E12 (Kanamycin resistance), 3. E.coli BW25113 PTTQ18 (Ampicillin resistance), 4. E.coli BW25113 (Δydco) + E.ferg benE PTTQ18 (Ampicillin resistance), 5. E.coli BW25113 + A1 (Ampicillin resistance), 6. E.coli BW25113 (Δydco) + Rhod 9 benE PTTQ18 (Ampicillin resistance), 7. E.coli BW25113 + R1 (Ampicillin resistance).
2. Overnights of each strain in LB= 5ml LB + single colonies picked from selective plates.
3. Grow cells to 0.2AU.
4. Add 0.1mM IPTG (0.5ul of 1M IPTG) to 5ml culture.
5. Bring cells to 0.1AU.
6. Dilute cells to 1:100 IN LB= 19.8ml LB + 200ul culture.
7. Add 0.1mM IPTG to 1:100 dilution.
8. PLATE:
   50ul LB into columns 2,3,4,6,7,8,10,11,12.
   100ul 250mM benzoate into columns 1,5,9.
   Serial dilute benzoate across plate.
   Row 8= LB into columns 1,2,3; LB + strains into columns 4,5,6,7,8,9,10.
   Add 50ul cells into each row (except H1/H2/H3).

### **MIC assays**

**E. coli BW25113 MIC in Benzoate and Catechol**1. Place 50ul LB into columns 2-10.
2. Place 50ul (+) control LB into column 12.
3. Place 100ul (-) control LB into column 11.
4. Place 100ul (2M) Benzoate into A1/B1/C1/D1.
5. Place 100ul (2M) Catechol into E1/F1/G1/H1.
6. Place 50ul cells into columns 1-10 and column 12.
7. Cover with breath-easy membrane.
8. Incubate at 38'C for 20 hrs.

**E. coli BW25113 knockout strains (+ inserted benABCD/E) MIC in 125/62.5/31.25/15.625mM benzoate (microtitre plate)**1. Strains= 1. E.coli BW25113 (WT), 2. E.coli BW25113 (Δydco) keoi:75E12 (Kanamycin resistance), 3. E.coli BW25113 PTTQ18 (Ampicillin resistance), 4. E.coli BW25113 (Δydco) + E.ferg benE PTTQ18 (Ampicillin resistance), 5. E.coli BW25113 + A1 (Ampicillin resistance), 6. E.coli BW25113 (Δydco) + Rhod 9 benE PTTQ18 (Ampicillin resistance), 7. E.coli BW25113 + R1 (Ampicillin resistance).
2. Overnights of each strain in LB= 5ml LB + single colonies picked from selective plates.
3. Grow cells to 0.2AU.
4. Add 0.1mM IPTG (0.5ul of 1M IPTG) to 5ml culture.
5. Bring cells to 0.1AU.
6. Dilute cells to 1:100 IN LB= 19.8ml LB + 200ul culture.
7. Add 0.1mM IPTG to 1:100 dilution.
8. PLATE:
   50ul LB into columns 2,3,4,6,7,8,10,11,12.
   100ul 250mM benzoate into columns 1,5,9.
   Serial dilute benzoate across plate.
   Row 8= LB into columns 1,2,3; LB + strains into columns 4,5,6,7,8,9,10.
   Add 50ul cells into each row (except H1/H2/H3).

**Cloning *benABCD***

### **DNA extraction**

*Acinetobacter baylyi ADP1* and *Rhodococcus wratislaviensis strain 9* DNA was extracted from live cells. Cell cultures were grown overnight in 5mL LB. 2x1.5mL of cell culture was taken from ADP1 and Strain 9 to give a total of 4 cell cultures. DNA was extracted from each of these cultures using the DNeasy Ultraclean Microbial Kit:- 1.8mL of cell culture was centrifuged for 30sec @ 10,000g- Supernatant was removed and cell pellet was resuspended with powerbead solution- Solution was transferred to powerbead tube and 50uL of SL solution was added- Tubes were secured to vortex at max speed for 10min- Tubes were centrifuged for 30sec at 10,000g- Supernatant was removed- DNA concentrations were measured using a nanodrop system and concentrations were calculated at:

              Strain 9 sample 1: 231.2ng/µL, Strain 9 sample 2: 268ng/µL, ADP1 sample 1: 235.8ng/µL, ADP1 sample 2: 186.4ng/µL

              ### **PCR**

              To amplify the target benABCD gene, primers were designed to allow DNA polymerase binding.

              Primers:

              - *R. strain 9* fwd: CTGATCTAGAtatgactgaaaccctgtac rvs: ATCGGCATGCcgagatctgtgggcag- *E.fergusonii* fwd: gtcaGGATCCtatgcaaaaaactct rvs: atgtTCTAGAttaccccagatcgc- *A. bayli ADP1* fwd: CTAGGGATCCTatgccacgtattcccgtc rvs: ACTGTCTAGACtcatccctgatcgcctcc

                    gDNA used:

                    - *E. Fergusonii*: 30ng/uL,- *R. strain 9*: 10ng/uL, this was gDNA that had been extracted and diluted to 10ng/uL

                        PCR mix:

                        | Constituent | ADP1 | St.9 | E.Ferg(1uL) | E.Ferg(control) | E.Ferg(0.5uL) |
                        | --- | --- | --- | --- | --- | --- |
                        | 5xBuffer | 10uL | 10uL | 10uL | 10uL | 10uL |
                        | Q5 high GC buffer | 10uL | 10uL | 10uL | 10uL | 10uL |
                        | dNTPs | 2uL | 2uL | 2uL | 2uL | 2uL |
                        | Fwd Primer | 2uL | 2uL | 2uL | 2uL | 2uL |
                        | Rvs Primer | 2uL | 2uL | 2uL | 2uL | 2uL |
                        | Q5 polyermase | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
                        | gDNA | 1uL | 1uL | 1uL | 0uL | 0.5uL |
                        | mQ | 22uL | 22uL | 22uL | 23uL | 22.5uL |
                        | Total Volume | 50uL | 50uL | 50uL | 50uL | 50uL |

                        The PCR was run with the following protocols:

                        - 95ºC for 2min- 95ºC for 20sec x35- 53ºC for 10sec x35- 72ºC for 1min52sec x35- 72ºC for 5min- 12ºC ∞

                                    A gel was prepared by mixing 10mL of 1% agrose with 2uL of DyeRed. This was poured into a gel mould and allowed to solidify.

                                    Gel (well) loading:

                                    - 1st lane: 4µL of E. ferg (1µL) mixed with 2µL of dye- 2nd lane: 4uL of E. Ferg (0.5µL) mixed with 2uL of dye- 3rd lane: 4uL of E. Ferg (control) mixed with 2uL of dye- 4th lane: 4uL of St. 9 mixed with 2uL of dye- 5th lane: 1µL 1KB+ ladder

                                              The Gel was run for 20min at 80V.

                                              ### **DNA purification**

                                              In order to purify the PCR products, a DNA purification was performed.

                                              PCR products were mixed with:

                                              - *E.fergosonii*- 60µl of PCR product (with loading buffer) combined with 300µl of PB buffer- *R. strain 9*- 80µl of PCR product (with loading buffer) combined with 400µl of PB buffer- *A. bayli ADP1*- 25µl of PCR product (with loading buffer) combined with 125µl of PB buffer

                                                    These samples were centrifuged for 1min @ 13.3g

                                                    Supernatant was discarded and the pellet was re-suspended in 750µl of PB buffer and placed into centrifuge column and centrifuged repeated. Supernatant discarded. 30µL of heated Elution buffer was placed in the centrifuge column and DNA was eluted.

                                                    ### **Digestion and ligation**

                                                    A digest was done on PCR products of *A. bayli ADP1* and *R. Strain 9*

                                                    | Contents | *R. srain 9 insert(1A)* | PTTQ18(1B) | *A. baylyi ADP1 insert(2A)* | PTTQ18(2B) |
                                                    | --- | --- | --- | --- | --- |
                                                    | XbaI | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
                                                    | SphI | 0.5uL | 0.5uL | 0uL | 0uL |
                                                    | BamHI | 0uL | 0uL | 0.5uL | 0.5uL |
                                                    | Cutsmart | 2uL | 2uL | 2uL | 2uL |
                                                    | DNA | 5uL | 0uL | 3uL | 0uL |
                                                    | PTTQ18 | 0uL | 10uL | 0uL | 10uL |
                                                    | MQ | 12uL | 7uL | 14uL | 7uL |

                                                    Concentrations:

                                                    - Plasmid (PTTQ18): 49.3ng/uL- *R. strain 9*: 113.4ng/uL- *A. baylyi ADP1*: 314 ng/uL

                                                          This digest was allowed to run for 2hrs in 37degree bath. Following this, 37mL of Rsap was added to the vector samples and allowed to sit in the 37degree bath for 20min. The B samples were then run on a gel and a gel extraction was performed on the corresponding band.

                                                          Concentrations following gel extraction:

                                                          - 1B: 4ng/uL- 2B: 9.3ng/ul

                                                              The A samples were cleaned up with the ZYMO DNA clean and concentrator kit. The concentrations were:

                                                              - 1A: 61.5ng/uL 1.83- 2A: 74ug/uL 1.89
                                                                  **Ligation reaction**

                                                                  An overnight ligation reaction was set up with the following concentrations:

                                                                  | Contents | St.9 | ADP1 |
                                                                  | --- | --- | --- |
                                                                  | DNA insert | 5.6uL(1A) | 4.6uL(1B) |
                                                                  | Plasmid | 7uL(2A) | 5.4(2B)uL |
                                                                  | Ligase | 2uL | 2uL |
                                                                  | Buffer | 2uL | 2uL |
                                                                  | MQ | 3.4 | 6uL |

                                                                  ### **Transformation**

                                                                  The PTTQ18 plasmid with ligated benABCD cluster from Rhod.9 and ADP1 was transformed into the E.coli strain GB09. GB09 was made competent by suspending GB09 sored in glycerol at -80 degrees into 2xmicrocentrifuge tubes with 1.4ml CaCl2 (cold) in each tube. This was allowed to sit for 20min ice. The cells were centrifuged at 10,000g for 1min and the supernatant was discarded. This process was repeated so the cells were washed with CaCl2 3 times. The competent cells were then suspended in 1.4mL LB media and 4uL of the cloned vector from St.9 and ADP1 was added into each tube respectively. This was allowed to sit on ice for 20minutes and then heat shocked in a 42degree water bath for 60seconds. The cells were then spread on agar plates with 15µl antibiotic (Ampicillin), 3µl IPTG, 45µl XGal per 30mL of agar.

                                                                  The plates were grown overnight and following day a colony PCR was performed to check if the digestion and ligation reactions correctly incorporated the benABCD cluster from Rhod.9 and ADP1 into PTTQ18.

                                                                  ### **Colony PCR**

                                                                  A colony PCR was used on the transformants that were made on the 29/09/2020. Promising colonies were selected (3 for ADPI and 8 for Rhod9) based on blue/white selection (Xgal).The PCR master mix was made up with:M13 fwd 6.6ul, M13 rvs 6.6ul, Buffer, 52.8ul, Mytech (polymerase) 6.6ul and MQ 185.8ml.
                                                                  The PCR reaction was set up with Mastermix plus a swab of colony or MQ for negative control and benABCD for postive control.

                                                                  The PCR reaction was set for:

                                                                  - 95C for 5min- 95C for 10sec x30- 55C for 10sec x30- 72C for 2min x30- 72C for 20min

                                                                            Following PCR, the PCR products were run through a 1% agrose gel using DyeRed.

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# Team:DeNovocastrians/Results

[Home](https://2020.igem.org/Team%3ADeNovocastrians)

Team

[The Team](https://2020.igem.org/Team%3ADeNovocastrians/Team)
[Attributions](https://2020.igem.org/Team%3ADeNovocastrians/Attributions)
[Collaborations](https://2020.igem.org/Team%3ADeNovocastrians/Collaborations)

Project

[Description](https://2020.igem.org/Team%3ADeNovocastrians/Description)
[Experiments](https://2020.igem.org/Team%3ADeNovocastrians/Experiments)
[Results](https://2020.igem.org/Team%3ADeNovocastrians/Results)
[Notebook](https://2020.igem.org/Team%3ADeNovocastrians/Notebook)
[Engineering](https://2020.igem.org/Team%3ADeNovocastrians/Engineering)
[Implementation](https://2020.igem.org/Team%3ADeNovocastrians/Implementation)
[Contribution](https://2020.igem.org/Team%3ADeNovocastrians/Contribution)

[Parts](https://2020.igem.org/Team%3ADeNovocastrians/Parts)
[Safety](https://2020.igem.org/Team%3ADeNovocastrians/Safety)
[Human Practices](https://2020.igem.org/Team%3ADeNovocastrians/Human_Practices)
[Education](https://2020.igem.org/Team%3ADeNovocastrians/Education)
[Poster](https://2020.igem.org/Team%3ADeNovocastrians/Poster)
[Awards](XXXXX)

**Results**

***benE***

The *benE* transporter gene was extracted and amplified from three bacterial sources. The *benE* gene from both *Rhodococcus strain 9* and *Acinetobacter baylyi ADP1* was obtained from genomic DNA. The *benE* from *Escherichia fergusonii* was ordered and synthesised from the IDT laboratory. Gene amplification was successfully completed through the use of PCR.
The amplified *benE* PCR products from *E. Fergusonii* and *Rhodococcus strain 9* were successfully cloned into the pTTQ18 plasmid.
The pTTQ18 vector with *benE* inserts were transformed into the *E.Coli* strain *BW25113* with the native benzene transporter knocked out (ΔydcO). The transformants were used in growth curve assays to assess the import ability of the *benE* transporter and can be seen in the figure below.

![](https://static.igem.org/mediawiki/2020/4/44/T--Denovocastrians--LB_curve.png)
*Figure 1. Growth Curve of transformed BW23115 strains in the presence of 15mM benzoate.*

The transformed *E. coli* with the *benE* gene were grown in the presence of 15mM benzoate. The *benE* transformants (with parts [BBa\_K3694027](http://parts.igem.org/Part%3ABBa_K3694027) and [BBa\_K3694028](http://parts.igem.org/Part%3ABBa_K3694028)) performed better than the *BW25113* ΔydcO control, indicating the transporter was functioning and importing benzoate from solution.

![](https://static.igem.org/mediawiki/2020/d/de/T--DeNovocastrians--HPLCbenzo.png)
*Figure 2. High power liquid chromatography analysis of benzoate concentration.*

High powered liquid chromatography (HPLC) was performed on the LB + benzoate growth curve samples used in the growth curve experiment. The cells were removed from the solution and analysis was undertaken on the concentration of benzoate left in solution. The *benE* transformants had less benzoate left in solution compared to the *BW25113* ΔydcO control. This confirms the [BBa\_K3694028](http://parts.igem.org/Part%3ABBa_K3694028) and [BBa\_K3694028](http://parts.igem.org/Part%3ABBa_K3694029) *benE* biobrick parts were successfully importing benzoate from solution into the cell.

![](https://static.igem.org/mediawiki/2020/3/3f/T--DeNovocastrians--Catechol.png)
*Figure 3. High power liquid chromatography analysis of catechol concentration.*

HPLC was also used to determine the presence of catechol of the growth curve solutions as it is the end-point of the benzene degradation pathway. There was confirmed to catechol in the transformant with the [BBa\_K3694029](http://parts.igem.org/Part%3ABBa_K3694029) part. This means that the *E. coli* were importing and degrading benzoate, further confirmation of our successful cloning and expression of our *benE* biobrick parts.

***benABCD***

The *benABCD* genes were extracted and amplified from *Rhodococcus strain 9* and *Acinetobacter baylyi ADP1* through PCR. The amplified PCR products were digested and ligated into the pTTQ18 plasmid. A digestion check suggested the pTTQ18::*benABCD*(*strain 9*) vector had successfully incorporated the gene insert. The pTTQ18::*benABCD*(*strain 9*) vector was transformed into the *E.coli* strain *BW25113*. However, the growth curve analysis undertaken on the transformed *BW25113* cultures found no degradation of benzoate. The pTTQ18::*benABCD*(*strain 9*) may have been incorrectly incorporated or mutated through the cloning process. Sequencing data would be required to ensure the *benABCD* gene is correctly cloned into the pTTQ18 plasmid.

**Biosensor**

Our initial attempts to join DNA fragments (fluorescent protein genes and benzene/catechol regulatory sequences) together for the construction of our biosensor were unsuccessful. However, by ordering new primers, increasing annealing temperature during PCR and using an optimisation protocol set by Hilgarth and Lanigan (2020), we joined some fragments together. We were able to join *catM* binding region to mCherry fluorescent gene, the *catM* gene to the *catM* promoter, and the *benM* gene to the*benM* binding region.
After this successful joining, we aimed to transform these constructed parts into organism for testing. Unfortunately, we were unable to produce viable transformed colonies and were therefore unable to perform any colony pcr to confirm the full construction of our biosensor.

![](https://static.igem.org/mediawiki/2020/1/16/T--DeNovocastrians--results.png)
*Figure 4. Biosensor fragments we successfully joined.*

**Future targets for *benABCDE* transformation**

We identified two potential insertion targets in: the marine organism *Synechocytis cyanobacteria*, and, the common terrestrial organism *Bacillus subtilis*. Neither species were able to grow in/degrade benzoate in their native states. In the image below: A) *B. subtilis* grew in control media (containing glucose) but was unable to grow in 10mM/100mM benzoate; B) *Synechocystis cyanobacteria* grew in control media (BG-11) but was unable to grow in 10mM/100mM benzoate. Therefore, they are potential targets for the insertion of the benABCDE cluster in future projects.

![](https://static.igem.org/mediawiki/2020/d/de/T--Denovocastrians--targets.png)
*Figure 5. (Left) B. subtilis exhibited no growth in benzoate. (Right) Synechocystis cyanobacteria showed no growth in benzoate*
**References**

**Hilgarth, R. S. and Lanigan, T. M.** (2020). Optimization of overlap extension PCR for efficient transgene construction. *MethodsX* **7**:100759.

![](https://static.igem.org/mediawiki/2020/1/17/T--DeNovocastrians--E.coliSuperHero.png)