iGEM 2022 — Team denovocastrians

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### Week 1 (18 - 24 April)

This week our team attempted to amplify the *benABCD* gene cluster and the *benE* gene from the gDNA of *Rhodococcus* sp. op-like and *Acinetobacter Baylii* ADP1 using primers from the 2020 iGEM DeNovocastrians. This achieved mixed results. We were able to get positives for the *benABCD* cluster in *A. baylii* ADP1 and *Rhodococcus* sp. op-like as well as positives for benE from *Rhodococcus* sp. op-like.
We then performed a gel extraction followed by a digest of both the genes and pTTQ, our first vector for this project. The genes were then ligated into the vector and transformed into *E. coli* GB05.

### Week 2 (25 April - 1 May)

This week our team ran a colony PCR to confirm last weeks transformations. The results of this PCR were negative for each colony. However, our team decided to move forward and attempt to confirm with a digest.

### Week 3 (2 - 8 May)

Unfortunately, due to complications with the loading dye and loading of the gels in general the results of the miniprep and digests were inconclusive. We decided to attempt to perform PCR on the remaining minipreps of our vectors in order to confirm if the genes had been succesfully inserted.
These also came back negative.

### Week 4 (9 - 15 May)

We decided to reattempt the cloning of the *ben* clusters and transporters from *A. baylii* ADP1 and *Rhodococcus* sp. op-like. This time we decided to use T5 exonuclease dependant assembly (TEDA) (Xia, Y., Li, K., Li, J., Wang, T., Gu, L. and Xun, L. (2019). T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis. Nucleic acids research 47, e15-e15.). We designed new primers for this and ordered them.

### Week 5 (16 - 22 May)

This week our third year students, Ella, Luke, Angus, and Claudia began to brainstorm individual streams that they could explore as a part of the placement for their undergraduate degrees.

### Week 6 (23 - 29 May)

The new primers arrived and our team followed the TEDA cloning protocol to insert the *benABCD* cluster as well as the *benE* transporter from *A. baylii* ADP1 and *Rhodococcus* sp. op-like into the pTTQ18 vector. These were then transformed into *E. coli*.

### Week 7 (30 May - 5 June)

This week our team explored fundraising methods in order to get ourselves to Paris for the Jamboree! We also explored an avenue for collaboration- a podcast involving other iGEM teams talking about both their projects and their experiences in the competition.

### Week 8 (6 - 12 June)

A colony PCR was performed on our transformants unfortunately they were all negative. We theorised this might be due to the cut site we used when digesting our vector. We decided to reattempt the cloning with an XbaI cut site instead.

### Week 9 (13 - 19 June)

This week we repeated the TEDA cloning using a new cutsite for the digest of the vector. On Friday we were able to use colony PCR to determine that this round of cloning worked!

### Week 10 ( 20 - 26 June)

This week we miniprepped our samples and digested them to confirm that our cloning worked. Upon discovering that the digests were positive we prepared the samples for sequencing to further confirm. Throughout this whole process we learned a lot, in particular that the *benABCD* cluster from *Rhodococcus* sp. op-like is difficult to amplify with PCR however, a high GC enhancer and touchdown PCR protocol seem to do the trick.
This week we also had a sponsorship meeting with Samsara, company working towards infiniteplasttic recycling. We learned a lot about taking something from concept through to product and how to scale up from research to commercial use.

### Week 11 (27 June - 3 July)

This week, the third years - Ella, Luke, Angus, and Claudia- prepared presentations to propose their streams of research for their placement projects.

### Week 12 (4 - 10 July)

On Monday the third years presented their proposals to the advisors- Joachim, Evan, Dan and Niki.
On Tuesday, glycerol stocks of *E. coli* GB05 and DH5a with the *benABCD* cluster and *benE* transporter from *A. Baylii* ADP1 and *Rhodococcus* sp. op-like.

### Week 13 (11 - 17 July)

There was a two week break of iGEM work over our holidays for team members and advisors to visit their families and take a break.

### Week 14 (18 - 24 July)

Break continues.

### Week 15 (25 - 31 July)

This week we caught back up with each other after the break. Ella and Naomi worked on a short presentation to give in their meetup with UT Austin. This meeting was to discuss where each team is at with their projects and discuss potential collaboration. Ella prepared questions to ask other teams who want to join a podcast.
Luke perfomred some preliminary investigation into what vessels would be used when growing bacteria in monoaromatic hydrocarbons.

### Week 16 (1 - 7 August)

This week Ella and Naomi adapted their presentation for UTAustin to also be given to the Australian Research Council Centre of Excellence in Synthetic Biology (ARCCoESB). Ella recieved an email from UPS informing the team that the distribution kit was on it's way.
Luke coninued his investigation into growth containment and performed volatility experiments to assess the retainment of monoaromatic hydrocarbons in bacterial growth media.

### Week 17 (8 - 14 August)

The meeting with UT Austin had to be rescheduled to next week. Naomi and Ella spoke in front of the ARCCoESB about the project.
Luke extracted genomic DNA from our novel benzene degrader (*Rhodococcus* sp. 33) for sequencing. However, the quality of this gDNA was not ideal for sequencing.

### Week 18 (15 - 21 August)

This week in the meeting with UT Austin we learned all about a fungus that affects the bats of Austin and how it can be addressed with Synthetic Biology. Ella and Angus ordered primers for qPCR of the *benABCD* cluster from *A. baylii* ADP1 and *Rhodococcus* sp. op-like.
Luke repeated gDNA extraction for our *Rhodococcus* strain. gDNA quantification showed that this sample was ideal for sequencing and the samples were sent off.

### Week 19 (22 - 28 August)

Ella miniprepped the pTTQ18 plasmid with the *benABCD* cluster from *A. baylii* ADP1 and *Rhodococcus* sp. op-like from *E. coli* GB05 and DH5a and transformed it into *E. coli* BL21. She then attempted to grow these organisms on a benzoate carbon source with a glucose control.
This week Luke ran the filming of our Project Promotion Video. He worked with Naomi and prepared the clips, scripts, assets and programs for video production.
Angus began the process of constructing the catechol responsive side of the biosensor. This began with using PCR to amplify a transcription factor and corresponding DNA binding region from *A. baylyi* ADP1 gDNA. PCR did not initially yield both fragments and gradient PCR was performed to troubleshoot annealing temperatures. Once the transcription factor and DNA binding region was successfully taken from *A. baylyi* ADP1 gDNA the biosensor plasmid was digested in preparation for insert of the amplified fragments.
Claudia ordered primers for for qPCR of the *benE* gene and HisTag insert. While waiting for primers to arrive she attempted direct site mutagenesis to remove a Bsai site from host plasmid pTTQ18 to create an GTG bsai empty vector. A diagnostic gel confirmed the removal of a BsaI site in pTTQ18 (pTTQRGSBsaI)

### Week 20 (29 August - 4 September)

Luke edited the promotional video for submission on the 2nd of september. This included creating animations, editing filmed content of the team, editing stock footage and narrating the script.
This week we also had a meeting with Samsara, an industry sponsor, in order to have a general chat about their work and ours while recording the first episode of the DeNovocastrians podcast.
Ella continued to attempt to grow *E. coli* expressing benzoate degradation genes in benzoate but was having no luck. She discussed using *E. coli* as a host organism as it is the parent strain of the Keio collection and the same strain that Claudia is working with. Unfortunately it's ability to grow on benzoate meant that an untransformed culture could not be used as a negative control. She also attempted to amplify the ben cluster from both gDNA and previous minipreps of pTTQ with the cluster insert.
Angus attempted TEDA cloning to insert the amplified fragments into the biosensor. This was initially unsuccessful and a number of troubleshooting steps proceeded.
Claudia's *benE* primers arrived and she sucessfully amplified the *benE* gDNA from *A. baylii* ADP1 via KOD Hot Start polymerase protocol. The amplification of *benE* from *Rhodococcus* sp. took multiple attempts however the addition of a high GC enhancer and Q5 high fidelity protocol preformed by Ella worked. Furthermore, a HisTag was added via TEDA cloning protocol to pTTQRGSBsaI using EcoR1 digested plasmid. The insertion of the HisTag was comfirmed via colony PCR (pTTQRGS6xHisBsaI).

### Week 21 (5 - 11 September)

Ella had a meeting with Evan where it was discussed that the reason there may be no growth on benzoate was the positioning of the ribosome binding site. They designed primers for recloning the *benABCD* cluster from *A. baylii* ADP1 and *Rhodococcus* sp. op-like with a correct ribosome binding site and into a new vector (pTTQRGS6xHisBsaI). These primers arrived at the end of week and Ella ran a PCR to amplify *benABCD, benA, benB, benC* and *benD* from *A. baylii* ADP1 and *Rhodococcus* sp. op-like. She was able to amplify all of them.
Angus continued troubleshooting cloning.
Luke performed growth experiments with our *Rhodoccocus* strain and discovered that it did not grow as expected in BTEX. Further investigation found that it required a different growth media. Luke also performed growth experiments with our *Rhodococcus* strain on more complex carbohydrates.
Claudia successfully transformed pTTQRGS6xHisBsaI into *E. coli* DH5α chassis.

### Week 22 (12 - 18 September)

Ella submitted the final safety form, with the edits suggested from HQ and finalised the team roster. Luke made a more complex growth media for use in BTEX growth experiments with our novel *Rhodococcus* strain. He also performed a growth experiment in BTEX. This was used to collect total protein samples for mass spectrometry analyses.
Angus attempted TEDA cloning once again after a number of attempts to troubleshoot the process. After another failure with TEDA cloning it was decided that Gibson Assembly would be used. Colony PCR was used to determine the success of Gibson Assembly. Colony PCR indicated a possible successful cloning. The colony was cultured and the plasmids purified. A diagnostic digest of the plasmid was performed with positive results. The biosensor now appeared to be successfully constructed and was sent for sequencing to confirm.
Claudia did a EcoR1 digest of the pTTQRGS6xHisBsaI plasmid and attempted to insert *benE* gDNA to form to variants of the plasmid. Cells where transformed into *E. coli* DH5α chassis.

### Week 23 (19 - 25 September)

Ella attempted to clone the *benABCD* cluster from *A. baylii* ADP1 and *Rhodococcus* sp. op-like into pTTQ18His and transformed the vector into *E. coli* DH5a. All of the colony PCRs showed negative results. It was theorised this was due to the digest being incomplete or due to incorrect annealing of overhangs. The pTTQRGS6xHisBsaI vector had an extra BsaI site in the *ampR* gene that had to be removed in order for the cloning to work properly. Ella miniprepped the plasmid from an overnight culture and used site directed mutagenesis in order to remove the extra BsaI site (Liu, H. and Naismith, J. H. (2008). An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC biotechnology 8, 1-10.). This was confirmed with a digest. Luke performed the final protein preparation for mass spectrometry, which included trypsin digestion and clean up. The protein samples collected and prepared from our *Rhodococcus* strain grown in BTEX were then sent off for mass spectrometry.
Claudia attempted colony PCR on the two *benE* pTTQRGS6xHisBsaI variants (*benE* *Rhod* pTTQRGS6xHisBsaI and *benE* ADP1 pTTQRGS6xHisBsa), however results were inconclusive.

### Week 24 (26 September - 2 October)

Ella finalised the project title and abstract along with the preliminary judging form. A meeting was scheduled for a podcast episode recording with the USYD iGEM team next Tuesday. Ella completed the cloning of *benABCD, benA, benB, benC* and *benD* from *A. baylii* ADP1 and *Rhodococcus* sp. op-like into pTTQRGS6xHisBsaI and transformed the vector into *E. coli* DH5a. Luke received raw data from mass spectrometry. Claudia proceded to do a diagnostic digest on the *benE* pTTQRGS6xHisBsaI plasmids, utilising a virtual gel as a predidiction guide. The diagnostic digestic yielded good results, almost completely matching the virtual gel. Only note is the EcoR1 double digest preformed for *benE Rhodococcus* variant showed different banding to what was predicted. We assume this is due to the promixity of the multicloning sites in both the insert and vector of that plasmid. Plasmids were sent for sequencing.
Angus began preparations for biosensor testing involving media preparations and making chemical stocks.

### Week 25 (3 - 9 October)

Distribution kit arrived. Ella performed colony PCRs along with minipreps and digests to confirm the cloning from last week. She was able to confirm the cloning of the cluster, *benA, benC*, and *benD* from *A. baylii* ADP1 and *benA, benC* and *benD* from *Rhodococcus* sp. op-like.
The team worked on the slides for their final presentation video. Luke worked on mass spectrometry analyses. Initial findings identified considerable upregulation (as well as downregulation) of proteins in our *Rhodococcus* strain in response to benzene. This will require further investication to identify upregulated proteins and propose their function in BTEX degradation.
Angus received the sequencing for the biosensor indicating that it had been successfully constructed as it was designed. Preliminary fluorescence testing of the biosensor in *Escherishia coli* DH5a was commenced. Testing consisted of measuring fluorescence in a microplate reader.
Claudia's plasmids were prepped for expression assays, testing in *E. coli* BW25113.

### Week 26 (10 - 12 October)

Angus continued testing of the biosensor in *Escherishia coli* BW25113 as this strain was deemed more suitable. Testing protocols were refined with the addition of a number of controls. Results from fluorescence tests were positive indicating the successful function of the biosensor.
Claudia started expression assays. Via a western blot Claudia showed expression for both *benE Rhod* pTTQRGS6xHisBsaI and *benE* ADP1 pTTQRGS6xHisBsaI variants via a western blot.
Ella completed expression experiments for her succesfully cloned strains, she then extracted the total soluble proteins and ran them on a gel to compare to a negative control.
Wiki freeze is today. This is the DeNovocastrians signing off.

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# Try, Try, and Try Again

When our team initially cloned the Ben clusters from *Acinetobacter baylyi* and *Rhodococcus* into the *E. coli* strain, the transformation was unable to use benzoate as a carbon source. At first, we tried different concentrations of benzoate in the media but there was still no growth. We went back to our original cloning process, and we noticed that the ribosome binding site was too far away from the start codon. Once we had noticed this, we redesigned our primers for PCR amplification and cloning so that this could be corrected. We have successfully cloned 6 genes from  *A. baylyi* and *Rhodococcus* Op-like and one full gene cluster from  *A. baylyi*. We had similar troubles when attempting to grow another *Rhodococcus* strain in minimal media with benzene as a carbon source. In this case however, we were able to follow a different method and use a new media to see growth on benzene as a carbon source.

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## *benE* Transporter

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*benE* transpoters amplified from two different organisms, *Rhodococcus* sp. and *A. baylyi* ADP1, were optimised with a His-tag in order to be visually expressed. The expression of the benE protein is shown by a western blot (Figure 1).

![Western Blot benE](https://static.igem.wiki/teams/4431/wiki/310826521-836067224062611-7748607569934006295-n.png)

**Figure 1. Lanes 1 & 2 are controls with no induction.** Lanes 3 & 5 are benE *Rhodococcus*sp. Lanes 4 & 6 are *benE* *A. baylyi*  ADP1.

## Biosensor

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In order to determine that the completed biosensor was functioning as expected a fluorescent assay was performed. The biosensor was transformed into *Escherichia coli* BW25113 and grown for approximately 20 hours in a CLARIOstar microplate reader and fluorescence data for GFP and mCherry was collected. Data was normalised against uninoculated media controls, meaning fluorescence readings are a result of biosensor activity.

![benzoate biosensor](https://static.igem.wiki/teams/4431/wiki/benzoate-biosensor.jpg)

**Figure 2. Fluorescent assay of GFP expression.** Black data points represent fluorescence in the presence of benzoate while grey represents no benzoate.

Data points are an average of n=3 replicates.

By analysing the data obtained from the fluorescence assay (Figure 2) we were able to determine that the addition of benzoate resulted in an increase in GFP fluorescence. We can therefore determine that the addition of benzoate results in an increase in GFP expression. Comparing the fluorescence of GFP in the presence of benzoate and without allows us to determine that the fluorescence is not just a result of an increasing cell density.

![catechol biosensor](https://static.igem.wiki/teams/4431/wiki/catechol-biosensor.jpg)

**Figure 3. Fluorescent assay of mCherry expression.** Black data points represent fluorescence in the presence of catechol while grey represents no catechol.

Data points are an average of n=3 replicates.

Similar results were seen when the fluorescence data for mCherry (Figure 3) was analysed. The addition of catechol resulted in an increase in mCherry fluorescence, which we determine to be a result of increased mCherry expression.

### Summary

We predict that the increases in fluorescent gene expression seen in this data are a result of upregulation by transcription factors which are responding positively to the presence of the relevant chemical (benzoate or catechol). More testing would be necessary to confirm that this is true.

## Cloning the *benABCD* genes

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Initially, we managed to clone the  *benABCD*  cluster from *A. baylyi*  and *Rhodococcus* sp. however, the ribosome binding site was too far from the start codon for us to expect efficient expression. Additionally, the vector used for this cloning was not pTTQRGS6xHisBsaI and thus, we redid the cloning of each cluster and the indivivual genes with new primers and the new vector.
Prior to the recloning an expression analysis was performed on BL21 transformed with the cluster from each organism. The total soluble and insoluble protein was extracted from pelleted cells and run on an acrylamide gel which is shown below. There are some differences in the protein bands however, further analysis is required to draw any firm conclusions.

![acrylamide gel of total protein](https://static.igem.wiki/teams/4431/wiki/bluebois.png)

The first step in cloning each of the genes and clusters into the new vector was to amplify the inserts. PCR was used for this with primers designed to introduce the necessary 5' overhang for TEDA cloning. In order to confirm correct amplification the purified PCR products of each of the individual genes was run on a gel as shown below. The PCR was performed in duplicate, with the first set of replicates on the left and the second set on the right. Each of the genes are separated by ladders i.e. *benA* from *A. baylii* is followed by *benA* from *Rhodococcus*, then a 1kb ladder, then *benB* from *A. baylii* and *benB* from *Rhodococcus* etc.
The bands for each of these genes are all of the expected sizes.

![PCR to amplify all genes](https://static.igem.wiki/teams/4431/wiki/initial-gene-pcr.png)

PCR was also used to amplify the entire cluster from each organism. The resulting products were also run on a gel to confirm.
TEDA cloning was then used to insert the amplified fragments into the pTTQRGS6xHisBsaI vector. The entire solution was used to transform *E. coli*  DH5a. Several colonies were picked for colony PCR in order to confirm the cloning and transformations. The results for *A. baylii benA, benB*, and *benD* and also *Rhodococcus benA* and the *benABCD* cluster were negative from both colonies picked, so four more colonies were chosen for colony PCR.
After both colony PCRs, there was at least one positive colony for each gene and cluster from both organisms.
Overnights were made up and the plasmids were miniprepped and digested in order to confirm the fragments had been inserted into the vector. This allowed us to confirm that the cloning had been correct for *A. baylii benA, benC, benD* and *benABCD* as well as for *Rhodococcus benA, benC* and *benD*. Some of the bands were difficult to make out on the initial gel, and so these were repeated with a lower voltage, longer runtime, and an EtBr stain. The results of both gels are shown below.

![Digest of plasmids initial](https://static.igem.wiki/teams/4431/wiki/digest-one.png)

![Digest of plasmids, repeated](https://static.igem.wiki/teams/4431/wiki/digest-two.png)

The miniprepped plasmids containing these genes were then used to transform colonies of E. coli BL21 for expression experiments.
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