iGEM 2019 — Team Victoria_Wellington

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

# Team:Victoria Wellington/Notebook

[Home](https://2019.igem.org/Team%3AVictoria_Wellington)

Team

Project

Lab

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

[Parts](https://2019.igem.org/Team%3AVictoria_Wellington/Parts)

[Judging Form](https://2019.igem.org/Team%3AVictoria_Wellington/Judging_Form)

[Members](https://2019.igem.org/Team%3AVictoria_Wellington/Members)

[Collaborations](https://2019.igem.org/Team%3AVictoria_Wellington/Collaborations)

[Attributions](https://2019.igem.org/Team%3AVictoria_Wellington/Attributions)

[Inspiration](https://2019.igem.org/Team%3AVictoria_Wellington/Inspiration)

[Description](https://2019.igem.org/Team%3AVictoria_Wellington/Description)

[Safety](https://2019.igem.org/Team%3AVictoria_Wellington/Safety)

[Timeline](https://2019.igem.org/Team%3AVictoria_Wellington/Timeline)

[Protocols](https://2019.igem.org/Team%3AVictoria_Wellington/Protocols)

[Results](https://2019.igem.org/Team%3AVictoria_Wellington/Results)

![](https://static.igem.org/mediawiki/2019/4/46/T--Victoria_Wellington--Logo.png)

# Team Victoria

# Notebook

April

May

June

July

August

September

October

X

## April

Cell Competency 18/04/2019

2x 400mL flasks with 1 in 100 dilution of overnight culture. Incubate at 37°C.

Grow until OD600 reaches 0.4-0.6.

Pour 1 flask worth of culture into pre-chilled 50mL Falcon tubes and ice for 20 minutes.

Spin at 3190rpm for 10 minutes, decant supernatant and add remaining culture on top of pellets.

Spin again

Add 50mL MQ over 4 tubes and resuspend pellets. Tip resuspended cultures into remaining 4 Falcon tubes and resuspend. Spin at 3190rpm for 10 minutes. Remove supernatant.

Add 50ml 10% glycerol to 2 tubes and resuspend pellets (down to 2 tubes). Spin at 3190rpm for 10 minutes.

Add 50mL to 1 tube, resuspend pellet, tip over remaining tube and resuspend pellet at 3190rpm for 15 minutes.

Tip off all supernatant. Allow residual glycerol drip down sides aand add additional 400mL of 10% glycerol. Fully resuspend final pellet with 1mL tipe. Should have ~1.1mL of super dense culture.

Freeze 85mL aliquots at 80°C.

Snap freeze in freeze block and test competency from frozen.

This was done using two E. coli strains: DH5α and BL12.

Carbon Paste Experiment 26/04/2019

Aim: Build electrodes of different graphite to oil ratios. Test their conductivity in salt solution

Method:

1. 2g of graphite was ground with 2mL of paraffin oil.

2. 1g of graphite was ground in a mortar and pestle with 2mL of paraffin oil.

Observations: The 1:1 ratio was a thick viscous paste. The 2:1 was near liquid in viscosity. It look like the 2:1 will not be able to support itself in the anode.

3. Not enough paste was obtained. So 4g of graphite to 4mL of paraffin was used for the 1:1 anode. 3g of graphite to 6g of paraffin was used for the 1:2 anode

Observations: The 1:2 anode was liquid and ran straight through. The 1:1 paste was a good consistency and could hold itself & wires in the tube.

4. 12g of NaCl was added to 100mL of distilled H2O to make a saturated NaCl (aq) solution.

5. A circuit was set up as below

6. The LED glowed proving the paste was conductive

![](/wiki/images/thumb/a/a0/T--Victoria_Wellington--Electrodes-Test.png/800px-T--Victoria_Wellington--Electrodes-Test.png)

Conclusion: From this experiment we proved that the electrodes were conductive and that 1:1 paste is a very good electrode!

However we found some issues trying to take accurate data past that.

Firstly, trying to fix the wire in place proved to be difficult. Due to this, we couldn't calculate the resistance of the CPE as the system would always be disturbed upon us plugging in the multimeter

![](/wiki/images/c/c9/T--Victoria_Wellington--Multimeter-Sketch.png)

In order to calculate the resistance we will need some way to immobilise the wire (using wax most likely) to get an accurate reading from the meter

## May

PCR (+ Prep) 01/05/2019

Alex prepared some agar, made more broth.

Made up 1x TE buffer.

Add 250µL to primers to bring up concentration to 100 pmol/µL.

Primers at 100 pmol/µL. Need to dilute further to be 0.5 µM = C = 0.5x10-6M

Currently:

C = 100 x 10-12 mol/µL-1.

C = 100 x 10-6 mol/L-1.

Dilution factor:

C2 = 𝑥/50µL x C1

0.5x10-6M = 𝑥/(50x10-6L)x 100x10-6M

𝑥/50x10-6L = 5x10-3

𝑥 = 2.5x10-7L

𝑥 = 0.25µL of primer

PCR Method (from Alistair):

Reaction:

2x master mix --> 10µL

10 fold diluted forward primer --> 1µL

10 fold diluted reverse primer --> 1µL

10 fold diluted template --> 1µL

7 µL PCR H2O

Method:

5 µL of forward primer was diluted in 45 µL of PCR H2O.

The same was done for the reverse primer.

5µL of template is added to 45 µL of PCR H2O.

Add all reagents minus template to PCR tubes.

Add template last, place in machine.

Miniprep of Plasmid 02/05/2019

Plasmid needs to be mini-prepped from bacteria for PCR

Method:

2 mL of cells were added to an Eppendorf tube (we had 4 tubes).

The tubes were centrifuged and liquid poured off.

Note: 2 mL can't fit into one Eppendorf tube. So add 1 mL, centrifuge, pour off unwanted liquid and then add 1 more mL.

Centrifuge at full speed for 1 minute.

Suspend pellet in 200µL of PDI.

Vortex the cells to mix thoroughly.

Add 200 µL of PD2.

If SDS is insoluble in PD2 (you can see crystals) add the bottle to a hot water bath for 5 minutes.

Invert the tube 4-6 times to mix. Note: after PD2 is added be gentle as PD2 makes DNA single-stranded so

it's now susceptible to mechanical damage.

Wait for 2 minutes.

Add 300µL of PD3.

Centrifuge for 5-10 minutes.

Pipette the clear liquid into Geneaid miniprep column (Plasmid sticks to the column).

Spin for 60 seconds

Pass the flow through back through the column and spin for a further 30 seconds.

Discard flow-through, return the catcher.

Add 400µL of W1, spin for 60 seconds and discard the flow-through.

Add 2x 300µL of wash to column. Spin for 60 seconds each time. Discard flow-through.

Spin for 2 minutes to remove CH3CH2OH.

Transfer column to fresh Eppendorf.

Add 100µL of 65°C EB as this removes DNA from column.

Let stand for 1-5 minutes.

Column can also be heated to 65°C to enhance elution.

Centrifuge for 1 minute.

Harvest DNA!

Initial nanodrop = 6.6ng/µL DNA.

Second/Refined = 13ng/µL DNA.

PCR 06/05/2019

Dilute primers 10-fold from 100µM to 10µM.

Used four PCR tubes, 1 control and 3x pET 28a +

In each reaction vessel:

20 µL Q5 master mix

1 µL Forward primer

1 µL Reverse primer

1.2 µL Template (plasmid)

Total reaction volume (µL) = 23.2

Agarose Gel Electrophoresis

Gel composition:

100 mL 1% agar mix

5 µL of ethidium bromide

Run for 30 minutes at 135 volts

## June

###### 1/6/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.

Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.

###### 2/6/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

## July

###### 1/7/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.

Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.

###### 2/7/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

## August

###### 1/8/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.

Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.

###### 2/8/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

## September

###### 1/9/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.

Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.

###### 2/9/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

## October

###### 1/10/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.

Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.

###### 2/10/2019

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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

# Team:Victoria Wellington/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://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3AVictoria_Wellington)
[HOME](https://2019.igem.org/Team%3AVictoria_Wellington)

TEAM

[Team Members](https://2019.igem.org/Team%3AVictoria_Wellington/Team)
[Collaborations](https://2019.igem.org/Team%3AVictoria_Wellington/Collaborations)

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AVictoria_Wellington/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AVictoria_Wellington/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AVictoria_Wellington/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AVictoria_Wellington/Hardware)
[Measurement](https://2019.igem.org/Team%3AVictoria_Wellington/Measurement)
[Model](https://2019.igem.org/Team%3AVictoria_Wellington/Model)
[Plant](https://2019.igem.org/Team%3AVictoria_Wellington/Plant)
[Software](https://2019.igem.org/Team%3AVictoria_Wellington/Software)

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

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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

# Team:Victoria Wellington/Results

[Home](https://2019.igem.org/Team%3AVictoria_Wellington)

Team

Project

Lab

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

[Parts](https://2019.igem.org/Team%3AVictoria_Wellington/Parts)

[Judging Form](https://2019.igem.org/Team%3AVictoria_Wellington/Judging_Form)

[Members](https://2019.igem.org/Team%3AVictoria_Wellington/Members)

[Collaborations](https://2019.igem.org/Team%3AVictoria_Wellington/Collaborations)

[Attributions](https://2019.igem.org/Team%3AVictoria_Wellington/Attributions)

[Inspiration](https://2019.igem.org/Team%3AVictoria_Wellington/Inspiration)

[Description](https://2019.igem.org/Team%3AVictoria_Wellington/Description)

[Safety](https://2019.igem.org/Team%3AVictoria_Wellington/Safety)

[Timeline](https://2019.igem.org/Team%3AVictoria_Wellington/Timeline)

[Protocols](https://2019.igem.org/Team%3AVictoria_Wellington/Protocols)

[Results](https://2019.igem.org/Team%3AVictoria_Wellington/Results)

![](https://static.igem.org/mediawiki/2019/4/46/T--Victoria_Wellington--Logo.png)

# Team Victoria

# Results

## Results Sections

### Aim

As we wanted to build an enzymatic fuel cell, a key component of our project is to characterise those enzymes - of particular interest were their kinetic parameters and pH optima.

The enzymes we tested were c. boidinii formate dehydrogenase and m. thermophila formate dehydrogenase. The main experiments conducted were as follows:

* Michaelis-menten kinetics of each enzyme with formate
* Optimum pH assay with each enzyme
* Crude kinetics of each enzyme with mesoxalate

### Methods

Since these enzymes use NAD+ as a cofactor, we were able to follow catalysis by examining increases in absorbance at 340nm with a spectrophotometer as NAD+ was reduced to NADH.

NADH was quantified in formate assays using a standard curve. This contained formate, phosphate buffer and a range of NADH concentrations. Using the resulting regression equation, NADH concentration (and quantity) could be calculated. Due to the reaction stoichiometry, we could infer that for each mole of NADH produced, 1 mole of formate had also reacted.

Due to the double-substrate nature of these enzymes' kinetics, there were additional considerations for the assay design. When measuring kinetics parameters for the substrate of interest (i.e. formate), its concentration is varied, but [NAD+] must be held constant.

Reaction conditions for each test were as follows:

* Formate kinetics - pH 7.5, 30℃, [NAD+] = 10mM, in 100mM phosphate buffer. Formate varied from 1-216mM
* pH assays - 30℃, [NAD+] = 10mM, [formate] = 200mM in 100mM phosphate buffer(s)
* Mesoxalate kinetics - pH 7.5, 20℃, [NAD+] = 5mM in 100mM phosphate buffer

Reactions were done in triplicate at 200𝜇l, warmed to temperature before adding enzyme and incubating in a thermocycler. Absorbance was read in a cuvette or 96-well plate.

Control reactions were included throughout; one without enzyme, one without NAD+ and one without formate. No enzyme controls were selected as blanks, and their absorbance subtracted from the other data.

For pH assays, as series of 100mM phosphate buffers at pH 6.5-8.5 were made up.

### Results

cbFDH kinetics

![](https://static.igem.org/mediawiki/2019/5/5c/T--Victoria_Wellington--Parts-cbFDH_mm.png)

A Michaelis-menten model was fitted in R, and kinetic parameters were calculated for cbFDH with formate. It’s residual standard error was 0.222

substrate

Vmax (μmol/min/mg)

Km (mM)

Kcat (min-1)

Specificity constant (M-1s-1)

Conditions

Formate

4.19

28.4

169.3

99.4

pH = 7.5, T = 30℃, 10mM NAD

Results were similar to those obtained by *Guo, Qi et al. [1]*

### pH assays:

Each FDH enzyme was tested at pH 6.5-8.5, and resulting data was plotted in R, along with a polynomial model fitted to each dataset:

![](https://static.igem.org/mediawiki/2019/c/c4/T--Victoria_Wellington--Parts-FDH_ph.png)

### mtFDH:

Equation: Activity = -1.06(pH)2 +17.5(pH) -65.6 (R-square = 0.94 - strong correlation)
Derivative: da/dpH = -2.12(pH) +17.5
Optimum pH = 8.26

### cbFDH:

Equation: Activity = -0.63(pH)2 +10.16(pH) -38.62 (R-square=0.96 - strong correlation)
Derivative = -1.26(pH) +10.16
Optimum pH = 8.06

The main finding here is that mtFDH has the best activity at a higher pH than cbFDH (even if only slightly).

It is important we acknowledge the shortfalls of this particular experiment. In each case, no substantial decrease in activity was observed, so a polynomial equation is not necessarily reflective of what happens outside the pH range we tested.

Of additional concern is that the cbFDH had been frozen longer than mtFDH before the assays were done. That mtFDH has a generally high activity here, is therefore not necessarily reflective of the enzyme’s properties - cbFDH is likely to have degraded.

Later kinetic assays also demonstrated mtFDH activity at pH 7.5 to be considerably lower - there appears to have been a systematic error in this experiment. We have not found an explanation to this, and as such the pH pattern, but not activity values, are the key outcome from these assays.

### mtFDH kinetics

An initial assay was done, and a Michaelis-Menten model fitted in R:

![](https://static.igem.org/mediawiki/2019/1/11/T--Victoria_Wellington--Parts-ftFDH1_mm.png)

Residual standard error was 0.273. Of note was that the activity shown here did not align at all with what was observed in the pH assay (for pH 7.5). As such, a repeat experiment was done:

![](https://static.igem.org/mediawiki/2019/a/a7/T--Victoria_Wellington--Parts-MM_ftFDH.png)

Again we found activity to be lower than in our pH assay. Due to the repetition from these kinetics assays, we decided these must be accurate.

The second model fitted had a residual standard error of 0.172 (< model 1) so this was selected for calculation of kinetic parameters:

substrate

Vmax (μmol/min/mg)

Km (mM)

Kcat (min-1)

Specificity constant (M-1s-1)

Conditions

Formate

4.53

32

197.2

102.8

pH = 7.5, T = 30℃, 10mM NAD

Overall, under the same reaction conditions mtFDH performed better than the cbFDH. It also seems to function better at high pH than cbFDH. Both these characteristics are desirable for our enzymatic fuel cell, and so if we were to select one enzyme it would be mtFDH.

One caveat with this conclusion is that cbFDH assays were done long after expression, so it may have degraded (which was not the case for mtFDH). Repeat experiments after defined amounts of time would be recommended.

### Mesoxalate kinetics:

![](https://static.igem.org/mediawiki/2019/0/0a/T--Victoria_Wellington--Parts-mesoxalate_FDH.png)

Although values are very small, we can still make qualitative conclusions from these data. Both formate dehydrogenases have a detectable reaction with mesoxalate - which is not their natural substrate. The mtFDH reacts more quickly than cbFDH, but as cbFDH had been idle in the freezer much longer, it is possible this is due to degradation.

### References

*[1] Guo, Qi et al. “Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii.” Biochemistry vol. 55,19 (2016): 2760-71. doi:10.1021/acs.biochem.6b00181*

iGEM Victoria 2019

[![facebook link](https://static.igem.org/mediawiki/2019/f/f4/T--Victoria_Wellington--facebook-mosaic.png)](https://www.facebook.com/iGEMVUW/)
[![twitter link](https://static.igem.org/mediawiki/2019/5/50/T--Victoria_Wellington--twitter-mosaic.png)](https://twitter.com/vuwsynbio)
![mail link](https://static.igem.org/mediawiki/2019/0/06/T--Victoria_Wellington--email-mosaic.png)