iGEM 2019 — Team Macquarie_Australia

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Macquarie Australia/Notebook

![](https://static.igem.org/mediawiki/2019/3/3c/Igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://static.igem.org/mediawiki/2019/a/a0/T--Macquarie_Australia--2019logo.jpeg)](https://2019.igem.org/Team%3AMacquarie_Australia)
[HyDRA:
Hydrogen Detection
for Real Applications](https://2019.igem.org/Team%3AMacquarie_Australia)

OUR TEAM

[Meet The Team](https://2019.igem.org/Team%3AMacquarie_Australia/Team)
[Collaborations](https://2019.igem.org/Team%3AMacquarie_Australia/Collaborations)

OUR PROJECT

[Description](https://2019.igem.org/Team%3AMacquarie_Australia/Description)
[Design](https://2019.igem.org/Team%3AMacquarie_Australia/Design)
[Experiments](https://2019.igem.org/Team%3AMacquarie_Australia/Experiments)
[Notebook](https://2019.igem.org/Team%3AMacquarie_Australia/Notebook)
[Characterisation](https://2019.igem.org/Team%3AMacquarie_Australia/Contribution)
[Results](https://2019.igem.org/Team%3AMacquarie_Australia/Results)
[Demonstrate](https://2019.igem.org/Team%3AMacquarie_Australia/Demonstrate)
[Improve](https://2019.igem.org/Team%3AMacquarie_Australia/Improve)
[Attributions](https://2019.igem.org/Team%3AMacquarie_Australia/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AMacquarie_Australia/Parts)
[Basic Parts](https://2019.igem.org/Team%3AMacquarie_Australia/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AMacquarie_Australia/Composite_Part)

[SAFETY FIRST!](https://2019.igem.org/Team%3AMacquarie_Australia/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AMacquarie_Australia/Human_Practices)
[Integrated Human Practices](https://2019.igem.org/Team%3AMacquarie_Australia/Integrated_Human_Practices)

AWARDS

[Best Composite Part](https://2019.igem.org/Team%3AMacquarie_Australia/Composite_Part)

[JUDGING FORM](https://igem.org/2019_Judging_Form?team=Macquarie_Australia)

# NOTEBOOK

Week 1 (02/07/19 - 04/07/19)

Dry Lab

* First team meeting was led by Ari Edmonds (2017 and 2018 Macquarie University iGEM team member), introducing synthetic biology and how the competition works. We met our advisors from the Department of Molecular Sciences and met Abigail Sison from iGEM HQ
* Professor Robert Willows discussed theoretical aspects of H2 biosensors and NiFe hydrogenases in *E. coli* which guided us in researching and reading literature
* We set up team accounts for Gmail, Facebook, Instagram and Twitter. We also brainstormed possible team names
* Eli and Sam brainstormed ways we can address human practices through a prototype

Wet Lab

* Anika, Daniel, Jerresa, Tasneem and Zoe prepared LB CAM and LB AMP plates. We transformed DH5ɑ with Plac, cyclic di-GMP phosphodiesterase, double terminator, RBS (parts from the 2019 iGEM distribution kit) in an attempt to build a component of our biosensor while we waited for synthesised parts to arrive

Week 2 (09/07/19 - 11/07/19)

Dry Lab

* As a team, we discussed logo ideas and potential contacts who can help to design and digitise our ideas.
* Alice, Carly and Eli drafted emails for human practices stakeholders, industry experts and sponsorship
* Anika, Carly, Daniel, Tasneem and Zoe brainstormed activity ideas for Macquarie University Open Day and developed an online survey based on which was shared to our social media pages
* Jerresa, Josip, Sam, Tasneem and Zoe researched methods for detecting cyclic-di-GMP.
* Each team member drafted their bios for the wiki.

Wet Lab

* Transformations of Plac, cyclic di-GMP phosphodiesterase, double terminator, RBS we performed last week was unsuccessful. Anika, Daniel, Jerresa, Tasneem and Zoe reattempted these transformations.
* We attempted to make our own competent *E. coli* cells.

Week 3 (16/07/19 - 18/07/19)

Dry Lab

* Everyone researched factors which may affect the functioning of cyclic-di-GMP phosphodiesterase.
* Eli and Sam received feedback from A/Prof Louise Brown regarding their prototype design,
* Alice and Tasneem received feedback from A/Prof Louise Brown regarding emails for human practices and sponsorship document
* Carly, Jerresa, Josip and Sam summarised the literature we read and highlighted important points and protocols we could use for our parts
* Ari showed us how to plan part construction using standard assembly and 3A assembly.

Wet Lab

* We learned how to prepare an agarose gel, use the NanoDrop and Gel Visualiser
* Anika, Daniel, Jerresa, Tasneem and Zoe miniprepped the plasmids containing Plac, cyclic di-GMP phosphodiesterase, double terminator, RBS then determined DNA concentration using the NanoDrop. We digested samples with *EcoRI, PstI* and *XbaI* then ran digests on a 1 % agarose gel.
* Anika, Daniel, Jerresa, Tasneem and Zoe completed standard assembly for Plac and RBS
* Anika, Daniel, Jerresa, Tasneem and Zoe completed 3A assembly for cyclic di-GMP phosphodiesterase, double terminator and TET backbone
* After digestion, we ran samples in a 1 % agarose gel and annotated bands

Week 4 (23/07/19 - 25/07/19)

Dry Lab

* Alice finalised our sponsorship document and emails for human practices and contacted 35+ companies and organisations
* We attended the Department of Molecular Sciences Masters of Research poster session and interacted with postgraduate research students and their supervisors. We noted what layouts and designs made posters stand out, ideas for our Jamboree poster
* Jerresa and Tasneem contacted Team Edinburgh UG and Team NU\_Kazakhstan for a possible collaboration as we are all working with hydrogen producing microbes
* Received our hydrogenase gblock from IDT so we started planning for digestion and ligation reactions
* Alice, Carly, Eli, Jerresa, Tasneem and Zoe attended the CSIRO ON Prime Finals at the Sydney Startup Hub to support the Macquarie Biohydrogen group
* We started preparing a presentation for our advisors summarising the work completed during our intensive month long iGEM bootcamp
* We received replies from Bondi Bioworks and Bioplatforms Australia, organised meetings for next week
* Alice and Eli attended a meeting with Andrew Gilbert from Bioplatforms Australia to discuss our project
* James Brown from Bondi Bioworks visited the team on campus to discuss entrepreneurship in synthetic biology and his expertise in the field

Wet Lab

* No colonies from our standard assembly and 3A assembly plates
* Digested hydrogenase gblock and KAN backbone with *EcoRI* and *PstI*, ligated then transformed. Colonies on transformed plates were inoculated to prepare liquid cultures
* Anika, Daniel, Eli, Jerresa, Tasneem, Sam and Zoe carried out miniprep of the phosphodiesterase, followed by digestion and ran samples on an agarose gel and SDS-PAGE

Week 5 (29/07/19 - 02/08/19)

Dry Lab

* First week of semester!
* Sam consulted our advisor, Russel, for planning our biofilm formation assay with the phosphodiesterase. Daniel, Jerresa and Sam also worked with our advisor, Dr Edward Moh to compare protocols for biofilm formation assays found in literature. We considered* parameters such as culture duration, dilution, media, incubation periods, wash steps and stains
  * We received a reply from Fire + Rescue NSW. Alice arranged a meeting with Paul Beylerian (Fire + Rescue NSW Scientific Officer) who will visit us on campus for an interview. We started preparing questions to ask and researched background information on Fire + Rescue NSW
  * We had our first weekly meeting with our advisors. Alice, Anika, Carly, Daniel, Eli, Josip, Sam and Tasneem presented a slideshow with our accomplishments throughout the iGEM bootcamp
  * We received our riboswitch with and without the terminator stem loop (RON and ROFF)
  * Alice and Eli met with Andrew Gilbert from Bioplatforms Australia to discuss our project

Wet Lab

* Jerresa and Sam ran an SDS-PAGE of YHJH in Lac and YHJH in KAN
* Sam prepared 5 liquid cultures of yhjh for the preliminary biofilm assay
* Started assembly of our riboswitch

Week 6 (05/08/19 - 09/08/19)

Dry Lab

* Our advisor, Dr Paul Jaschke, ordered the hydrogenase component of our biosensor from Twist
* Alice contacted various high schools who may be interested in hearing about our project
* Alice, Zoe, A/Prof Louise Brown and Sam King attended an interview with Paul Beylerian from Fire + Rescue NSW. We developed a better understanding of the limitations of current gas detectors used by firefighters
* We received our first silver sponsorship from Labcyte!
* Jerresa presented the weekly update presentation to our advisors
* Hannah joined our iGEM team! She is completing a Masters of Research and plans to help us with laboratory work and writing literature reviews for the wiki.

Wet Lab

* Sam checked the preliminary biofilm assay for the yhjh part and performed the wash step with acetic acid and OD readings.
* Anika transformed the ligated RON and ROFF plasmids in DH5α though plates had no colonies.
* Carly, Daniel, Eli, Josip, Sam, Tasneem and Zoe reattempted the transformation of RON and ROFF
* Two biofilm assays were prepared. One plate will be incubated at 28 °C, the other incubated at room temperature (kept in the drawer).
* Jerresa prepared overnight cultures of RON and ROFF transformations. Anika, Jerresa, Daniel, Tasneem and Zoe miniprepped these transformations which was followed by digestion. Jerresa and Tasneem ran an agarose gel of the digested RON and ROFF samples.

Week 7 (12/08/19 - 16/08/19)

Dry Lab

* Alice, Carly, Daniel, Jerresa, Tasneem and Zoe have volunteered to represent our team at the Macquarie University Open Day. We started discussing ideas about engaging ways to interact with visitors and prospective students relating to our project.
* With the help of our advisor, Dr Roy Walker, we contacted Team Edinburgh for a possible collaboration as they are using the 2017 Macquarie iGEM team’s construct.
* Carly, Josip and Tasneem participated in a Skype call with Team NTU Singapore to discuss a possible collaboration. For the past 3 years, Macquarie Australia and NTU Singapore have collaborated by performing qPCR and SDS-PAGE for parts. We decided it would be great to continue this collaborative relationship.
* Alice started preparing presentations for high school visits
* Carly finalised our iGEM poster for the Macquarie University Open Day
* The team presented the weekly update presentation to our advisors
* Alice and Jerresa attended an interview with Macquarie University PhD recipient, Dr Vanessa Pirotta. Dr Vanessa is experienced in science communication and has a strong passion for sharing science with non-scientific audiences. She discussed how we can engage with our social media followers, gave advice on creating presentations and taught us ways to communicate complex information in inspiring and relatable terms
* Alice drafted a second round of emails for human practices and sponsorships based on the feedback given by Dr Vanessa Pirotta
* Alice, Ari, Carly, Daniel, Jerresa, Tasneem and Zoe represented the team at the Macquarie University Open Day. Our stall was located next to the teaching laboratories where the tours ended. This gave us the opportunity to educate tour groups about iGEM and how we plan to utilise synthetic biology to address issues. Ari demonstrated a hydrogen fuel cell toy car and we encouraged visitors to complete our short survey

Wet Lab

* Daniel, Tasneem and Zoe repeated the agarose gel due to diffusion of the samples and distorted bands
* Josip and Sam prepared digestion of the enzymes, *PstI, SpeI, XbaI* and *EcoRI* to check if they were still usable. Jerresa and Zoe ran a 1 % agarose gel of the digests
* Sam prepared the next biofilm formation assay.

Week 8 (19/08/19 - 24/08/19)

Dry Lab

* We took headshots for team introductions on our social media pages
* The team presented the weekly update presentation to our advisors
* We were getting inconsistent results from our agarose gels. After consultation with Professor Robert Willows, we concluded that it may be due to the activity of our enzymes. Thi decided to order new enzymes (*EcoRI, PstI, XbaI, SpeI, NdeI*)

Wet Lab

* Carly, Eli and Hannah digested the riboswitch with *EcoRI* (single) and *PstI* (double). Eli and Zoe ran an agarose gel of the digests. The gel did not appear to run properly (agarose didn’t melt, samples were not loaded properly) so it was repeated
* Sam took the OD readings of last week’s biofilm formation assay and followed suggestions from our advisor, Dominic, regarding the wash step and measurement

Week 9 (26/08/19 - 30/08/19)

Dry Lab

* We received replies from Hazer Group and Maximator, organised phone call interviews for next week
* Carly continued planning a collaboration with Team NTU Singapore
* Carly, Jerresa and Ari participated in a Skype call with Team NU\_Kazahkstan to discuss our projects and possible ways we could collaborate
* We received a platinum sponsorship from Bioplatforms Australia and a bronze sponsorship the Macquarie University Student Representative Committee!
* Our advisor, Samuel King, organised a human practices meeting with Alice, Anika, Carly and Daniel to discuss ways to address the medal criteria
* Alice, Daniel, Jerresa and Zoe presented our project to high school students at Brigidine College, St Ives
* Alice and Jerresa volunteered to represent the team at the Synthetic Biology Australasia Conference in Brisbane in October. They started planning flights, accommodation and registration with the guidance of A/Prof Louise Brown, Anwar Sunna and Louise Burton (CSIRO)
* Josip ordered riboswitches with inducible promoters (lac and tac)
* The team presented the weekly update presentation to our advisors

Wet Lab

* We made observations for RON and ROFF plates
* According to a paper found by Sam, *E. coli* Nissle 1917 is more efficient in producing biofilms compared to DH5α. We obtained these cells from our advisors, Dr Paul Jaschke and Dominic Logel, which were used to make competent cells
* We decided to rebuild the cyclic-di-GMP phosphodiesterase plasmid

Week 10 (02/09/19 - 07/09/19)

Dry Lab

* Selected photos from the Brigidine school presentation to upload to our social media pages
* Jerresa, Sam and Zoe presented the weekly update presentation to our advisors
* Daniel started designing our team shirts and received feedback from our advisors
* Ari, Daniel, Jerresa, Sam and Zoe participated in a photoshoot for the Faculty of Science and Engineering
* We were approached by Team UAAAN to collaborate and planned a video call for next week
* Alice started drafting the abstract for the wiki and Synthetic Biology Australasia Conference

Wet Lab

* Hannah retransformed the PDE in Nissle 1917 with the following changes to our protocol
* We sent sent RON and ROFF samples for sequencing
* Hannah performed miniprep of PDE and miniprep of yhjh KAN and CAM
* Hannah also helped prepare overnight liquid culture for cyclic-di-GMP and RON CAM and ROFF CAM
* With the guidance of Professor Robert Willows, we prepared a preliminary GFP fluorescence assay using our riboswitch. Unfortunately, when we tested variables (growth media, temperature), the plate dried out and results were highly variable.
  ![Aleq](https://static.igem.org/mediawiki/2019/c/cc/T--Macquarie_Australia--NotebookGFPprelim.jpeg)

Week 11 (09/09/19 - 13/09/19)

Dry Lab

* Received our hydrogenase (Hyd 1A and 1B) and riboswitches with promoters. Josip started planning assembly of these parts
* Carly, Tasneem and Sam presented the weekly update presentation to our advisors. During our meeting, we finalised the SBA and wiki abstract with feedback from advisors
* Alice, Carly, Sam, Eli, Tasneem, Zoe, Hannah, Jerresa and Josip discussed gel image from 09/09 with Thi and Ed. When overnight cultures were made, two colonies may have been selected or incomplete digestion. Alternatively, colonies were not transformants and possible contamination from the miniprep kit.
* Ed, Thi, Carly and Jerresa participated in a video call with NTU Singapore. We hope to send our riboswitch after sequencing confirmation so they can perform qPCR. They will send us a cell lysate and nuclear pellets for protein expression and SDS-PAGE
* Carly and Jerresa participated in a Skype call with Team UAAAN to discuss possible ways we could collaborate
* Carly, Jerresa, A/Prof Louise Brown, Prof Robert Willows and Macquarie biohydrogen group participated in a phone call interview with Mark Edwards (Hazer Group)
* Jerresa and A/Prof Louise Brown submitted the team roster, abstract and title, safety form and track selection
* Received a reply from USYD for collaboration, arranging a Skype call for next Monday
* Alice prepared questions for the BOC interview which Carly sent and confirmed our meeting next Monday

Wet Lab

* Josip and Tasneem used the NanoDrop to find DNA concentration of yhjh, RON CAM, ROFF CAM, cyclic-di-GMP minipreps from 06/09
* Daniel performed an agarose gel to check the hydrogenase and cyclic-di-GMP
* Hannah, Josip and Zoe prepared an assay for our riboswitch with different salt concentrations
* Josip prepared liquid culture from transformant colonies from TACON, TACOFF, LACON, LAC OFF plates

Week 12 (16/09/19 - 20/09/19)

Dry Lab

* Alice, Carly, Jerresa, Sam, Zoe, Professor Robert Willows, Samuel King, Dr Tony Jerkovic attended a meeting with Chris Dolman from BOC, Australia's leading gas & safety equipment specialist
* Eli, Josip, Jerresa, Thi and Dr Paul Jaschke planned for Gibson Assembly of hydrogenase (Twist)
* Alice, Carly, Eli and Jerresa worked on prototype design and feedback from interviews
* Carly and Jerresa participated in video call with Team Sydney Australia. We decided the best way to collaborate would be performing an SDS-PAGE for one of their proteins, and in return, replicate one of our GFP assays. We organised a time to exchange samples
* Carly and Jerresa also arranged a video call with Team UAAAN for next week
* Anika worked on the UNSW Symposium slideshow
* Alice, Carly and Jerresa worked on content for the wiki
* Carly and Tasneem presented the weekly update presentation to our advisors. We also did a team selection process for Boston where members pitched their contributions, team achievements and what they hope to achieve from attending the Jamboree. Carly, Eli, Jerresa, Sam, Tasneem and Zoe applied.

Wet Lab

* Josip and Hannah performed digestion of Hyd 1A and 1B, cyclic-di-GMP using enzyme E for single digestion and a combination of E and N for the double digestion
* Hannah prepared competent cells of Nissle and DH5α
* Daniel and Anika performed digestion of Hyd and cyclic-di-GMP PDE
* Eli conducted a Gibson Assembly with the hydrogenase fragments and KAN backbone, which was transformed by Tasneem in DH5α
* Anika, Daniel, Sam and Tasneem restreaked colonies from Hydrogenase and CDG plates plus positive and negative controls
* Anika, Daniel, Hannah and Zoe made 2 agarose gels for Tac ON/OFF; Lac ON/OFF, Hydrogenase digests and CDG PCR, W and W/O digests and annotated both gels
* Jerresa prepared samples prepared for sequencing (CDG W KAN 3, CDG W/O CAM 2, Lac ROFF 1, Lac ROFF 2, Lac RON 1, Lac RON 2, Lac RON 4
* Jerresa and Sam transformed PDE with DH5α (commercial and homemade)
* Sam performed a Polymerase Chain Reaction (PCR) for the CAM backbone, KAN backbone and colony.
* Carly, Jerresa and Zoe made LB media and plates (LB CAM and LB KAN)
* Eli and Tasneem performed retransformation of Hyd 1A and Hyd 1B
* Sam made Congo Red plates for the spot assay
* Sam prepared a Congo red spot plate
* Hannah prepared glycerol stocks of Lac ROFF red colonies CAM, Lac ROFF green colonies CAM, RON CAM 1 DH5α, ROFF CAM 1 DH5α
* Hannah, Jerresa, Sam GFP Assay of Constitutive and Inducible Riboswitches with LB

Week 13 (23/09/19 - 27/09/19)

Dry Lab

* The team presented the weekly update presentation to our advisors
* Carly and Jerresa visited Team Sydney Australia at their campus to exchange samples for our collaborations
* Daniel worked on our shirt design
* Jerresa drafted content for the wiki

Wet Lab

* We started using our new Qiaprep spin miniprep kit (250). Miniprep and digestion of PDE + RON/OFF
* Anika performed a digestion of the hydrogenase and Tasneem ran an agarose gel of the digests SDS-PAGE of hydrogenase
* Hannah and Zoe prepared glycerol stocks for LacON KAN 2, LacOFF KAN 2, RON CAM, TacOFF KAN 3, TacON KAN 1, LacOFF KAN 1, LacON KAN 1, TacOFF KAN 2
* Josip prepared sequencing reactions for CDG W, CDG W/O, Hyd 1A, PDE, RON, ROFF, LACON, PDE + RON CAM 2, PDE + RON CAM 4, PDE + ROFF CAM 1
* Hannah prepared overnight cultures for competent cells
* Jerresa, Hannah, Russel prepared a GFP assay for the inducible and constitutive riboswitches with salt concentrations

Week 14 (30/09/19 - 04/10/19)

Dry Lab

* Alice and Jerresa presented the weekly update presentation to our advisors and practiced their presentation for the Synthetic Biology Australasia Conference
* Alice and Jerresa continued working on the SBA presentation with feedback from Associate Professor Louise Brown
* Alice worked on content for the wiki
* Eli, Josip, Sam, Tasneem, Thi, Dr Edward Moh and Professor Robert Willows discussed the spot assay results

Wet Lab

* Josip transformed Hyd A + B colonies 4 and 7, CDG W and CDG W/O, Nissle CDG W KAN, Nissle CDG W/O CAM, DH5α A+B CAM 4, DH5α A+B CAM 7. BL21 (DE3) cells were used for transformation of Team Sydney Australia’s *PsiD* gene.
* Anika, Daniel, Eli, Tasneem and Zoe performed an SDS-PAGE of the transformed PsiD (whole cell lysate, soluble fraction and pellet). Gel was repeated by Hannah
* Sam prepared a biofilm formation assay to test the yhjh (with and without the promoter and terminator) in DH5α and Nissle 1917, yhjh + RON/ROFF in DH5α and Nissle 1917
* Anika and Zoe performed agarose gel of digests and 3xGibson assembly
* Jerresa prepared a GFP assay for the yhjh + RON/ROFF samples
* Hannah and Josip miniprepped the CDG and yhjh
* Jerresa transformed PDE + RON/ROFF CAM with Nissle 1917

Week 15 (07/10/19 - 13/10/19)

Dry Lab

* We assigned team members to each wiki page and descriptions for the special prizes on the judging form
* Everyone worked on content for the wiki and descriptions for the judging form
* The team presented the weekly update presentation to our advisors

Wet Lab

* Jerresa prepared a GFP assay for yhjh + RON/ROFF with sucrose

Week 16 (13/10/19 - 20/10/19)

Dry Lab

* Alice and Jerresa travelled to Brisbane to attend the Synthetic Biology Australasia Conference and present our project to the synthetic biology community
* Eli, Sam, Tasneem and Zoe worked on the wiki and judging form
* Hannah wrote a report for the hydrogenase part
* Jerresa presented the weekly advisors meeting. We received feedback on how to write our results and content for the wiki
* All team members worked hard to finalise and upload content to the wiki and registry pages

Wet Lab

* Sam and Tasneem redid the Congo red plates and Biofilm assay
* Daniel performed SDS-PAGE for the Team NTU Singapore collaboration
* We assembled the small subunit, large subunit, protease and phosphodiesterase via 3A assembly.

Week 17 (21/10/19 - 22/10/19)

Dry Lab

* All team members continued to work hard to finalise and upload content to the wiki and registry pages. Go Team HyDRA!

Wet Lab

* Hannah and Jerresa performed an agarose gel for sequence confirmed parts. The last gel for iGEM 2019!


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Macquarie Australia/Experiments

![](https://static.igem.org/mediawiki/2019/3/3c/Igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://static.igem.org/mediawiki/2019/a/a0/T--Macquarie_Australia--2019logo.jpeg)](https://2019.igem.org/Team%3AMacquarie_Australia)
[HyDRA:
Hydrogen Detection
for Real Applications](https://2019.igem.org/Team%3AMacquarie_Australia)

OUR TEAM

[Meet The Team](https://2019.igem.org/Team%3AMacquarie_Australia/Team)
[Collaborations](https://2019.igem.org/Team%3AMacquarie_Australia/Collaborations)

OUR PROJECT

[Description](https://2019.igem.org/Team%3AMacquarie_Australia/Description)
[Design](https://2019.igem.org/Team%3AMacquarie_Australia/Design)
[Experiments](https://2019.igem.org/Team%3AMacquarie_Australia/Experiments)
[Notebook](https://2019.igem.org/Team%3AMacquarie_Australia/Notebook)
[Characterisation](https://2019.igem.org/Team%3AMacquarie_Australia/Contribution)
[Results](https://2019.igem.org/Team%3AMacquarie_Australia/Results)
[Demonstrate](https://2019.igem.org/Team%3AMacquarie_Australia/Demonstrate)
[Improve](https://2019.igem.org/Team%3AMacquarie_Australia/Improve)
[Attributions](https://2019.igem.org/Team%3AMacquarie_Australia/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AMacquarie_Australia/Parts)
[Basic Parts](https://2019.igem.org/Team%3AMacquarie_Australia/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AMacquarie_Australia/Composite_Part)

[SAFETY FIRST!](https://2019.igem.org/Team%3AMacquarie_Australia/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AMacquarie_Australia/Human_Practices)
[Integrated Human Practices](https://2019.igem.org/Team%3AMacquarie_Australia/Integrated_Human_Practices)

AWARDS

[Best Composite Part](https://2019.igem.org/Team%3AMacquarie_Australia/Composite_Part)

[JUDGING FORM](https://igem.org/2019_Judging_Form?team=Macquarie_Australia)

# Protocols

### Cells

 [Competent Cells](https://static.igem.org/mediawiki/2019/2/2e/T--Macquarie_Australia--competentcells.pdf)

### Cloning

 [3A Assembly](https://static.igem.org/mediawiki/2019/e/ec/T--Macquarie_Australia--3AAssembly.pdf)
 [Gibson Assembly](https://static.igem.org/mediawiki/2019/b/b7/T--Macquarie_Australia--gibsonassembly.pdf)
 [Chemical Transformation](https://static.igem.org/mediawiki/2019/a/a8/T--Macquarie_Australia--Chemicaltransformation.pdf)
 [Electroporation](https://static.igem.org/mediawiki/2019/e/e1/T--Macquarie_Australia--ElectroporationProtocol.pdf)
 [Miniprep](https://static.igem.org/mediawiki/2019/5/5e/T--Macquarie_Australia--miniprep.pdf)
 [Restriction Digestion](https://static.igem.org/mediawiki/2019/2/2c/T--Macquarie_Australia--restrictiondigest.pdf)
 [Agarose Gel Electrophoresis](https://static.igem.org/mediawiki/2019/9/90/T--Macquarie_Australia--Agaroseg.pdf)
 [Gel Extraction](https://static.igem.org/mediawiki/2019/b/b0/T--Macquarie_Australia--gelextraction.pdf)
 [SDS-Page](https://static.igem.org/mediawiki/2019/0/03/T--Macquarie_Australia--sdspage.pdf)

### Assays to Check Functionality

 [Induction](https://static.igem.org/mediawiki/2019/3/3c/T--Macquarie_Australia--Induction.pdf)
 [Biofilm Assay](https://static.igem.org/mediawiki/2019/2/28/T--Macquarie_Australia--Biofilmassay.pdf)
 [Spot Plate Assay](https://static.igem.org/mediawiki/2019/f/ff/T--Macquarie_Australia--congoredspotplate.pdf)
 [GFP Fluorescence Measurement](https://static.igem.org/mediawiki/2019/c/cb/T--Macquarie_Australia--GFPassay001.pdf)
 [Hydrogen Gas Detection](https://static.igem.org/mediawiki/2019/e/e8/T--Macquarie_Australia--ClarkElectrode.pdf)

Sponsors

![](https://static.igem.org/mediawiki/2019/3/37/T--Macquarie_Australia--sponsors.jpeg)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Macquarie Australia/Results

![](https://static.igem.org/mediawiki/2019/3/3c/Igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://static.igem.org/mediawiki/2019/a/a0/T--Macquarie_Australia--2019logo.jpeg)](https://2019.igem.org/Team%3AMacquarie_Australia)
[HyDRA:
Hydrogen Detection
for Real Applications](https://2019.igem.org/Team%3AMacquarie_Australia)

OUR TEAM

[Meet The Team](https://2019.igem.org/Team%3AMacquarie_Australia/Team)
[Collaborations](https://2019.igem.org/Team%3AMacquarie_Australia/Collaborations)

OUR PROJECT

[Description](https://2019.igem.org/Team%3AMacquarie_Australia/Description)
[Design](https://2019.igem.org/Team%3AMacquarie_Australia/Design)
[Experiments](https://2019.igem.org/Team%3AMacquarie_Australia/Experiments)
[Notebook](https://2019.igem.org/Team%3AMacquarie_Australia/Notebook)
[Characterisation](https://2019.igem.org/Team%3AMacquarie_Australia/Contribution)
[Results](https://2019.igem.org/Team%3AMacquarie_Australia/Results)
[Demonstrate](https://2019.igem.org/Team%3AMacquarie_Australia/Demonstrate)
[Improve](https://2019.igem.org/Team%3AMacquarie_Australia/Improve)
[Attributions](https://2019.igem.org/Team%3AMacquarie_Australia/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AMacquarie_Australia/Parts)
[Basic Parts](https://2019.igem.org/Team%3AMacquarie_Australia/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AMacquarie_Australia/Composite_Part)

[SAFETY FIRST!](https://2019.igem.org/Team%3AMacquarie_Australia/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AMacquarie_Australia/Human_Practices)
[Integrated Human Practices](https://2019.igem.org/Team%3AMacquarie_Australia/Integrated_Human_Practices)

AWARDS

[Best Composite Part](https://2019.igem.org/Team%3AMacquarie_Australia/Composite_Part)

[JUDGING FORM](https://igem.org/2019_Judging_Form?team=Macquarie_Australia)

# RESULTS

##### Riboswitch

Our biosensor utilises a riboswitch to control the expression of enhanced green fluorescent protein (eGFP) through the modulation of intracellular cyclic-di-GMP concentration. This regulation occurs at the transcription level. As the riboswitch/eGFP is being transcribed by the RNA polymerase, cyclic-di-GMP can bind to the aptamer region of the riboswitch (Fig. 1), which will cause the formation of a hairpin terminator loop structure, preventing the transcription of the downstream eGFP gene.

![](https://static.igem.org/mediawiki/2019/1/12/T--Macquarie_Australia--Riboswitch_Map.png)***Figure 1**: BioBrick structure of the riboswitch/eGFP operon. The cyclic-di-GMP binds at the riboswitch aptamer, forming the terminator structure and preventing the transcription of the eGFP region.*

In order to test the function of our riboswitch, we developed two riboswitch/eGFP operon constructs; one containing the terminator loop region within the riboswitch and one without. We hypothesised that the construct containing the terminator stem structure would constitutively express eGFP, regardless of cyclic-di-GMP concentration. Because of this constant activity, we refer to this construct as “R ON” (for riboswitch ON) [[BBa\_K3151002]](http://parts.igem.org/Part%3ABBa_K3151002). Alternatively, the construct with the terminator stem would have the expression of eGFP be repressed by native cellular concentrations of cyclic-di-GMP, with this construct being referred to as “R OFF” (for riboswitch OFF) [[BBa\_K3151011]](http://parts.igem.org/Part%3ABBa_K3151011).

![](https://static.igem.org/mediawiki/2019/9/99/T--Macquarie_Australia--ribocartoon.png)***Figure 2**: The hypothesised activity of the two riboswitch constructs. While the RNA polymerase (red) moves along the operon (blue), cyclic-di-GMP (green) will bind to the aptamer region of the riboswitch transcript (black). When bound to cyclic-di-GMP, the R OFF construct **(Fig. 2.1a)** will produce the terminator loop structure **(Fig. 2.1b)** and cease transcription, preventing the expression of eGFP. When cyclic-di-GMP binds to the R ON transcript **(Fig. 2.2a)** it does not form the terminator loop structure **(Fig 2.2b)**, thereby having no effect on the expression of eGFP.*

We ordered these sequences from Twist Bioscience and assembled them via BioBrick standard assembly. Assembly confirmation was performed using restriction digestion with EcoRI and PstI (Fig. 3) and sequencing (Macrogen). These constructs were transformed into *E. coli* DH5α.

![](https://static.igem.org/mediawiki/2019/6/66/T--Macquarie_Australia--THEFINALGEL.png)

***Figure 3**: Agarose gel of single and double digests of sequence confirmed parts.*

The promoter selected for this construct is a stationary phase promoter [[BBa\_J45992]](http://parts.igem.org/Part%3ABBa_J45992), which was selected because cyclic-di-GMP concentration varies with cell growth cycles, with its highest level of expression being as it enters stationary phase[1]. We also tested the function of the constructs with two inducible promoters; Lac [[BBa\_R0010]](http://parts.igem.org/Part%3ABBa_R0010) and Tac [[BBa\_K180000]](http://parts.igem.org/Part%3ABBa_K180000) to characterise the efficacy of our promoter.

We collaborated with  [Team NTU-Singapore](https://2019.igem.org/Team%3ANTU-Singapore) (Nanyang Technological University) to perform Quantitative Polymerase Chain Reaction (qPCR) to measure the rate of transcription of eGFP between the R ON and R OFF constructs under the control of the Lac promoter (Fig. 4). These results demonstrate that between 4 and 24 hours, when the cells are entering stationary phase, the increased concentration of cyclic-di-GMP results in a significant decrease in the transcription of the eGFP sequence in the R OFF transformants.

![](https://static.igem.org/mediawiki/2019/f/f2/T--Macquarie_Australia--lacqpcr.png)

***Figure 4**: qPCR of R ON and R OFF constructs expressed in *E. coli* DH5ɑ under the Lac promoter. Samples were induced with 0.5 mM IPTG. The forward and reverse primers used for the qPCR both lay within the eGFP sequence.*

In order to characterise the mode of action of the riboswitch/promoter combinations, we collaborated with  [Team Sydney Australia](https://2019.igem.org/Team%3ASydney_Australia) to perform a GFP fluorescence assay[2]. Samples were measured with the BMG Pherastar plate reader to measure eGFP (Ex485 nm Em520 nm) and OD (600 nm). These results demonstrate that the Lac and Tac promoters have significantly higher background expression of the R OFF construct.

This is likely due to the strength of the promoters, and the length of the transcripts produced by either construct. This is demonstrated clearly by the results of the Tac promoter **(Fig. 6 & 9)**, in which by the end of the assay the R OFF samples were producing higher levels of eGFP. We believe that this is due to the fact that the binding of cyclic-di-GMP to the transcript results in shorter transcripts, allowing the RNA polymerase to produce more of the transcripts quickly and saturating the cyclic-di-GMP, resulting in a loss of inhibition of eGFP transcription. This effect can also be observed in the samples under control of the Lac promoter **(Fig. 5 & 8)**. By contrast, the background level of transcription under the stationary phase promoter remains constant throughout the entire assay **(Fig. 7)**, making it a more suitable candidate for our biosensor.

|  |  |  |
| --- | --- | --- |
| ![Aleq](https://static.igem.org/mediawiki/2019/5/50/T--Macquarie_Australia--lac01.jpg) | ![Aleq](https://static.igem.org/mediawiki/2019/9/99/T--Macquarie_Australia--tac01.jpg) | ![Aleq](https://static.igem.org/mediawiki/2019/0/06/T--Macquarie_Australia--stat01.jpg) |
| ***Figure 5***: eGFP produced by cells transformed with the Lac riboswitch constructs over 60 hours. | ***Figure 6**: eGFP produced by cells transformed with the Tac riboswitch constructs over 60 hours.* | ***Figure 7**: eGFP produced by cells transformed with the Stationary phase riboswitch constructs over 60 hours.* |

|  |  |
| --- | --- |
| ![Aleq](https://static.igem.org/mediawiki/2019/7/78/T--Macquarie_Australia--UsydLac01.jpg) | ![Aleq](https://static.igem.org/mediawiki/2019/1/1c/T--Macquarie_Australia--UsydTac01.jpg) |
| ***Figure 8**: eGFP produced by cells transformed with the Lac riboswitch constructs over 30 hours. Data provided by Usyd iGEM.* | ***Figure 9**: eGFP produced by cells transformed with the Tac riboswitch constructs over 30 hours. Data provided by Usyd iGEM.* |

##### Phosphodiesterase

Cyclic-di-GMP acts as the secondary messenger within our detect-response system. It prevents the response (eGFP production) by binding to the terminator stem of the riboswitch. The hydrogenase we are utilising as our sensor contains a phosphodiesterase domain, which is modulated by the presence of molecular hydrogen. This allows it to digest cyclic-di-GMP, reducing the cellular concentration and allowing for the production of eGFP.

![](https://static.igem.org/mediawiki/2019/e/ed/T--Macquarie_Australia--hydact01.jpeg)

***Figure 10**: Hypothetical mode of action of the sense/response system of our biosensor. The hydrogenase will have its phosphodiesterase domain activated in the presence of molecular hydrogen. This will lead to a decrease in intracellular cyclic-di-GMP, preventing the riboswitch from forming the terminator loop structure and allowing the transcription of eGFP.*

In order to test this functionality, we assembled a plasmid containing a well characterised cyclic-di-GMP phosphodiesterase, yhjH [[BBa\_K861090]](http://parts.igem.org/Part%3ABBa_K861090), with an inducible Lac promoter [[BBa\_R0010]](http://parts.igem.org/Part%3ABBa_R0010) to create a new composite part [[BBa\_K3151008]](http://parts.igem.org/Part%3ABBa_K3151008). We also assembled a plasmid with just the *yhjH* gene with no promoter and terminator as a control. We ordered these sequences from Integrated DNA Technologies and assembled into BioBricks using standard assembly. Assembly was confirmed through restriction digestion **(Fig. 3)** and sequencing. These constructs were then transformed into the *E. coli* DH5α and Nissle 1917.

As intracellular cyclic-di-GMP concentration is directly correlated with biofilm formation[2], we can utilise the biofilm forming capability of the transformants as a proxy for intracellular cyclic-di-GMP concentration and phosphodiesterase activity. Nissle 1917 *E. coli* cells transformed with these constructs were spotted onto LB agar plates supplemented with Congo Red and Coomassie Blue[3], and the cells transformed with the construct containing the promoter and terminator sequences produced significantly less cellulose than cells transformed with the construct without the promoter and terminator, as well as parental Nissle 1917 cells (Fig. 11), indicating significant phosphodiesterase activity within the cell.

![](https://static.igem.org/mediawiki/2019/f/f4/T--Macquarie_Australia--yhjh1.png)

***Figure 11**: *E. coli* Nissle 1917 cells transformed with the yhjH phosphodiesterase with (A) and without (B) promoter and terminator, as well as negative control (C). Cells were spotted onto LB agar plates supplemented with Congo Red and Coomassie Blue, and visualised with Blue Light LEDs. Nissle 1917 was selected as it produces significantly higher levels of cellulose in its biofilm. The red colour observed in (B) and (C) is the Congo Red staining the cellulose.*

*E. coli* DH5α cells transformed with these same constructs were used to perform a microtitre plate adherence capacity assay**[X]**, another measure of the cells’ capacity for forming biofilms. This assay was particularly prone to error as the Crystal Violet used to stain the biofilm was particularly difficult to remove entirely, and extraneous stain increased the absorbance reading (Fig. 12).

![](https://static.igem.org/mediawiki/2019/c/ce/T--Macquarie_Australia--biofilm1.png)

***Figure 12**: Results from adherence capacity assay with *E. coli* DH5α transformed with the yhjH phosphodiesterase w/ and w/o promoter and terminator, as well as negative control parental cells.*

This *yhjH* phosphodiesterase has been used in a previous composite part [[BBa\_K2471001]](http://parts.igem.org/Part%3ABBa_K2471001), which utilised the T7 promoter for its expression. However, as we were not planning on assembling our constructs into cells with T7 RNA polymerase, as well as wishing to have inducible expression of our parts, we decided to improve upon the design of the previous BioBrick to better suit out purposes. We transformed both our Lac *yhjH* BioBrick as well at the T7 *yhjH* BioBrick into the *E. coli* strains DH5a, Nissle 1917 and BL21(DE3). These transformants were then spotted onto LB plates supplemented with Congo Red and Coommassie Blue in order to assay for their biofilm production capability **(Fig. 13)**.

The spot assay shows a significant reduction in cellulose synthesis in Nissle 1917 cells transformed with the Lac+yhjH compared with negative control, and little to no reduction in cells transformed with the T7+yhjH (Fig. 13). No significant change was seen in any of the DH5a cells (Fig. 15). No significant difference in colony morphology was observed between negative control BL21(DE3) and BL21(DE3) cells transformed with T7+yhjH, though some reduction in Congo-Red staining was observed in BL21(DE3) cells transformed with Lac-yhjH (Fig. 14), which indicates a reduction in cellulose biosynthesis and therefore an increase in phosphodiesterase activity.

![](https://static.igem.org/mediawiki/2019/3/35/T--Macquarie_Australia--NissleT7Lac.jpg)

***Figure 13**: Spot Assay of the negative control Nissle 1917 (A), Nissle 1917 with T7+yhjH (B), and Nissle 1917 with Lac+yhjH (induced with 1 mM IPTG) (C). Plate observed under blue light to enhance visualisation of Congo Red-stained cellulose.*

![](https://static.igem.org/mediawiki/2019/6/62/T--Macquarie_Australia--BL21DE3T7Lac.png)

***Figure 14**: Spot Assay of the negative control BL21(DE3) (A), BL21(DE3) with T7+yhjH (B), and BL21(DE3) with Lac+yhjH (induced with 1 mM IPTG) (C). Plate observed under white light to enhance visualisation of colony morphology.*

![](https://static.igem.org/mediawiki/2019/f/ff/T--Macquarie_Australia--DH5aT7Lac.png)

***Figure 15**: Spot Assay of the negative control DH5a (A), DH5a with T7+yhjH (B), and DH5a with Lac+yhjH (induced with 1 mM IPTG) (C). Plate observed under white light to enhance visualisation of colony morphology.*

## Phosphodiesterase and Riboswitch

In order to characterise the interaction between the function of our riboswitch and phosphodiesterases, we constructed two plasmids containing the coding sequences both the riboswitch/eGFP operon (either R ON and R OFF) under the stationary phase promoter and the *yhjH* gene under the Lac promoter, referred to as *yhjH* + RON [[BBa\_K3151029]](http://parts.igem.org/Part%3ABBa_K3151029) and *yhjH* + ROFF [[BBa\_K3151030]](http://parts.igem.org/Part%3ABBa_K3151030) respectively. We confirmed the assembly through restriction digestion as well as sequence confirmation. These plasmids were then transformed into the *E. coli* strains DH5α and Nissle 1917.

*E. coli* DH5α cells transformed with these plasmids we used to perform fluorescence assays using the BMG Pherastar plate reader to measure eGFP (Ex485 nm Em520 nm) and OD (600 nm). These results (Fig. 16) confirm our hypothesis that the ROFF construct will produce significantly lower levels of eGFP due to inhibition from cellular cyclic-di-GMP.

![](https://static.igem.org/mediawiki/2019/2/28/T--Macquarie_Australia--yhjhribo03.jpg)

***Figure 16**: The constitutive riboswitch [[BBa\_K3151011]](http://parts.igem.org/Part%3ABBa_K3151011) was expressed in DH5ɑ cells. The black lines represent R ON samples, the blue lines represent R OFF samples. The increased phosphodiesterase activity in the ROFF samples allowed the eGFP to be expressed at higher than baseline levels, though still significantly lower than RON samples.*

Nissle 1917 *E. coli* cells transformed these plasmids were spotted onto LB agar plates supplemented with Congo Red and Coomassie Blue. We compared the effect of induction of the yhjH phosphodiesterase between the yhjH+R ON and yhjH+R OFF transformants. Induction of yhjH appeared to have no effect on the fluorescence of yhjH+R ON transformants **(Fig. 17A, Fig. 17C)**, which corroborates our hypothesis that its expression of eGFP is not influenced by the intracellular concentration of cyclic-di-GMP. Induction of yhjH appeared to significantly increase the fluorescence of yhjH+R OFF transformants **(Fig. 17B, Fig. 17D)**, again corroborating our hypothesis that a decrease in intracellular cyclic-di-GMP will allow for an increase in expression of eGFP.

![](https://static.igem.org/mediawiki/2019/5/55/T--Macquarie_Australia--yhjhribo04.jpg)

***Figure 17**: LB agar plate supplemented with Congo Red and Coomassie blue, spotted with yhjH+R ON induced (A), yhjH+R OFF induced (B), yhjH+R ON uninduced (C), and yhjH+R OFF uninduced (D).*

##### Hydrogenase

We ordered hydrogenase constructs pUC57Kan-HydA (Hydrogenase large and small subunits, maturation protease) and pUC57Amp-HydB (cyclic-di-GMP phosphodiesterase) from GeneWiz. They were transformed into *E. coli* DH5α competent cells and amplified. To confirm the presence of the two parts, we performed single and double restriction enzyme digestions, followed by agarose gel electrophoresis and SDS-PAGE. The results were consistent with our expectations **(Fig. 18–20)**.

For pUC57Kan-HydA **(Fig. 18)**, we observed a single band at about 6000 bp from single digestion, representing the linearised plasmid DNA; for double digestion, a band at 3300 bp represented HydA, and the two bands at 900 bp and 1500 bp corresponded to the cleaved pUC57Kan.

![](https://static.igem.org/mediawiki/2019/6/61/T--Macquarie_Australia--hydfig1.png)

***Figure 18:** Agarose gel electrophoresis profiles of *Magnetospirillum magneticum* [NiFe] hydrogenase operon upstream part HydA. Four biological replicates were analysed.*

SDS-PAGE results **(Fig. 19)** showed distinct protein bands at approximately 17 kDa in two biological replicates, which corresponded to the maturation protease HupD **(Sample 4 and 7, Fig. 18b)**. As the hydrogenase operon was organised in a sequential order of *hurS, hurL,* followed by *hupD*, the expression of HupD suggested that both *hurS* and *hurL* should have been transcribed and translated into proteins.

![](https://static.igem.org/mediawiki/2019/8/88/T--Macquarie_Australia--hydfig2.png)

***Figure 19:** SDS-PAGE profiles of *Magnetospirillum magneticum* [NiFe] hydrogenase operon upstream part HydA. Four biological replicates were analysed.*

For double digestion of pUC57Amp-HydB, the bands at 2500 bp and 3000 bp represented pUC57Amp and HydB, respectively. Single digestion of pUC57Amp-HydB gave an intense band at approximately 3500 bp, which was the uncut circular plasmid. (Fig.20).

![](https://static.igem.org/mediawiki/2019/2/23/T--Macquarie_Australia--hydfig3.png)

***Figure 20:** Agarose gel electrophoresis profile of *Magnetospirillum magneticum* [NiFe] hydrogenase operon downstream part HydB. Four biological replicates were analysed.*

To test the functionality of the hydrogenase part HydA of *M. magneticum* [NiFe] hydrogenase, we measured the hydrogen saturation and desaturation rates (mV/s) in water, HydA7 (DH5ɑ +hydrogenase) and DH5ɑ (-hydrogenase) using a Clark-type electrode sensor. HydA7 was used for the functionality test, because it has the highest level of protein over-expression. The results showed a significant difference between the DH5ɑ and HydA7 bacterial cells in their rates of hydrogen absorption.

The hydrogen saturation and desaturation rates (**Fig. 21 and 22**), are represented as mV per second. The saturation rate in water was the highest, followed by HydA7 and then DH5ɑ (**Fig. 21**). Similarly, the desaturation rate in water was the highest, followed by HydA7 and the lowest, DH5ɑ (**Fig. 22**). The three samples represented in each graph are water, DH5ɑ (-hydrogenase), and (DH5ɑ +hydrogenase).

![](https://static.igem.org/mediawiki/2019/b/b5/T--Macquarie_Australia--hydfig7.png)

***Figure 21:** Maximum hydrogen saturation rates of water (standard), DH5α (negative control), and DH5α HydA7 transformant. Error bars are +/- StDev, n=2.*

![](https://static.igem.org/mediawiki/2019/8/8d/T--Macquarie_Australia--hydfig4.png)

***Figure 22:** Maximum hydrogen desaturation rates of water (standard), DH5α (negative control), and DH5α HydA7 transformant. Error bars are +/- StDev, n=2.*

Water is the standard, representing the maximum hydrogen saturation rates. When comparing the DH5ɑ with and without hydrogenase, a clear difference is demonstrated in the maximum rate of H2 saturation (mV/s; **Fig. 21**). H2 rate of consumption is higher in our HydA7 sample compared to untransformed DH5ɑ cells. There is overexpression of hydrogenase in the DH5ɑ HydA7 sample, therefore, in the presence of hydrogen, the HydA7 cells appeared to bind hydrogen, thus allowing for more hydrogen to be absorbed into solution before its maximum saturation is reached. This may be specifically attributed to the hydrogen binding in the hydrogenase active site. Conversely, the DH5ɑ cells without the hydrogenase did not appear to absorb as much hydrogen, resulting in lower hydrogen saturation rate. This result demonstrates that there is significant hydrogen binding in hydrogenase binding site of our modified HydA7 cells

A clear difference when comparing the DH5ɑ with and without hydrogenase is demonstrated once more with rate of H2 desaturation. When comparing the data for DH5ɑ (-hydrogenase) and our DH5ɑ (+hydrogenase), the desaturation rate for the HydA7 is higher compared to that of the DH5ɑ cells without the hydrogenase, demonstrating the ability of the hydrogenase to bind hydrogen.

Hydrogenases are known to be reversible, where they can both produce H2 or Detect H2. In order to demonstrate that our hydrogenase ([[BBa\_K3151025]](http://parts.igem.org/Part%3ABBa_K3151025)) does not function as a H2 producer, we performed a hydrogen electrode test **(Fig. 23)** comparing it to the hydrogen producing gene cluster (HPGC). This gene cluster was taken from the Macquarie\_Australia iGEM 2017 team. In black, is represented by *E. coli* (+HPGC) and in blue, is represented by *E. coli* (+Hydrogen sensor). The clear indication of hydrogen gas production can be seen by the HPGC, as opposed to the HydA7 (Hydrogen Sensor), which shows no indication that any hydrogen gas is being produced. This result shows that our [NiFe] hydrogenase taken from *M. magneticum* operates only in the direction of oxidizing hydrogen, and that the oxidation is not reversible.

![](https://static.igem.org/mediawiki/2019/f/f6/T--Macquarie_Australia--hydrogenproduction.jpg)

***Figure 23:** Hydrogen production in DH5α HydA7 transformant following addition on 20 mM glucose.*

##### References

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2. Dell EJ. Bottom Reading of Cell-Based Assays: Direct Optic Approach Enhances Fluorescent Protein and Other Microplate Analyses. Genetic Engineering & Biotechnology News. 2012 May 1;32(9):22-3.
3. Stepanović S, Vuković D, Ježek P, Pavlović M, Švabic-Vlahović M. Influence of dynamic conditions on biofilm formation by staphylococci. European Journal of Clinical Microbiology and Infectious Diseases. 2001 Jul 1;20(7):502-4.
4. Cimdins A, Simm R, Li F, Lüthje P, Thorell K, Sjöling Å, Brauner A, Römling U. Alterations of c‐di‐GMP turnover proteins modulate semi‐constitutive rdar biofilm formation in commensal and uropathogenic Escherichia coli. MicrobiologyOpen. 2017 Oct;6(5):e00508.
5. Ogasawara H, Yamada K, Kori A, Yamamoto K, Ishihama A. Regulation of the *Escherichia coli* csgD promoter: interplay between five transcription factors. Microbiology. 2010 Aug 1;156(8):2470-83.