Team:Cambridge-JIC/Collaborations

CAMBRIDGE-JIC

Collaborations

Glasgow Team

We can see how you fluoresce

Collaboration with the Glasgow iGEM Team was based on fluorescence imaging: as part of their project, the Glasgow team are characterising different fluorescence constructs expressing RFP and GFP. It was therefore beneficial for them to have an independent group analyse the bacterial expression of their constructs, and for us to have some living fluorescent samples to image. Specifically, the bacterial transformants they supplied were reported to have the following phenotypes:

  • DH5α - No antibiotic resistance, no fluorescence (control)

  • DH5α - Kanamycin resistance, GFP and RFP fluorescence (in the same cell)

  • DH5α - Kanamycin and Chloramphenicol resistance, RFP fluorescence

  • DH5α - Kanamycin and Chloramphenicol resistance, GFP fluorescence

  • DH5α - Kanamycin and Chloramphenicol resistance, mostly GFP fluorescence with a small proportion of cells on the plate express RFP

Our objective was therefore twofold: firstly to test the capabilities of OpenScope in analysing bacterial samples for GFP and RFP fluorescence, and secondly to report back to Glasgow with our phenotypic analysis of their bacterial strains.

William and Mary Team

As part of their project, the William and Mary iGEM Team were imaging cells expressing a variety of fluorescent protein including RFP, CFP, GFP, and YFP. The constructs are being used for the Interlab Measurement study. As part of the measurement track the W&M team assembled fluorescent constructs, and measured and reported their fluorescence. The samples were sent to us to confirm the correct assembly of the parts by validating their fluorescence. Their interest in seeing how their constructs performed (in terms of florescent output) perfectly matched our own need to test OpenScope on cell samples rather than fluorescent beads. In addition, optical components were obtained to extend our imaging capabilities to both GFP and RFP. In particular, RFP imaging has not been tested previously.

The W&M team were kind enough to send us dried DNA samples containing constructs for GFP and RFP expression in E. coli. Details on the protocols used to resuspend the DNA and to transform the cells can be found below.


Transformation Protocol for William and Mary iGEM Team 2015


Context

  • Using E. Coli K12 Dh5 alpha cells

  • Dry plasmid for two different GFP constructs in duplicate (so 4 samples total) sent on filter paper

  • All constructs on Amp resistance backbones pSB1C3

  • Constructs are J23106 + I13504 on pSB1C3, and J23117 + I13504 on pSB1C3

  • Constructs generated through DNA synthesizing the insert (J231XX + I13504) and using Gibson Assembly to insert it into the Amp backbone


Objective:

  • Transform cells with the constructs provided

  • Image the cells for RFP and GFP fluorescence using OpenScope to confirm functionality of the inserts, and the microscope itself


DNA Extraction:

Adapted from: Extraction protocol

  1. Cut center of circles out of the filter paper

  2. Added 50 µl of EB buffer to resuspend DNA

  3. Pipetted up and down to mix


Transformation:

Adapted from: Transformation protocol

  1. Add 1 µl of DNA extract to thawed out competent Top10 E. coli cells

  2. Mixture allowed to stand on ice for 15 minutes

  3. Cells heat shocked for 30 seconds at 42˚

  4. Mixture allowed to stand on ice for 5 minutes

  5. 100 µl of LB added at room temperature

  6. Cells incubated for 1hr at 37˚ with shaking

  7. Cells plated out on Amp plates containing rich medium


Results:

Conclusions:

UK Teams Meetup (Organised by Westminster Team)

Bringing microscopy to iGEMmers

In order for UK iGEM teams to meet, socialise and discuss their projects ahead of the Jamboree, the Westminster iGEM team organised a two day meeting in London. As the Hardware track was new to this year’s competition, and being the only team following this track in the UK, we felt this would be a fantastic opportunity to introduce other teams to this track and to highlight the important role of hardware in synthetic biology.
Our workshop took the form of three stations, each covering a slightly different aspect of our project and each with a different discussion focus:

Optics Bench

A working set-up of our first optics bench, and demonstration 3D printed material. Discussion points:

  • 3D printing and rapid prototyping

  • Principles of open-source hardware

  • Inverting the Raspberry Pi camera lens

  • Field of view and resolution calculation

Microscopy Awaits

A working set-up of our latest microscope prototype, brightfield and fluorescence modes, fully motorised and video streaming to the Webshell. Discussion points:

  • Importance of illumination, and difficulty with

  • Remote control over Webshell

  • Epi-cube explanation

  • Stage flex-principle demonstration

Build Your Own

An interactive demonstration on building our microscope from scratch. Discussion points:

  • Importance of documentation and instructions

  • Design choices

  • Discuss improvements required

  • How to use Arduino and Raspberry Pi


Images courtesy if the Westminster iGEM Team

The entire team was involved, and this allowed members of other teams to chat and ask questions at all three stations while the equipment was being run. The level of enthusiasm from other team members was extremely encouraging, and we were very surprised by the number of questions about the hardware track in general. We hope that as a result of our worksop, some teams may consider it for next year’s competition.
Perhaps one of the most important outcomes of the workshop was in raising awareness of the role of open source hardware (OSH). We emphasised the degree of collaboration and community that it fosters, in the context of our own project. Underlying this is the ability of other iGEM teams to build, modify and improve our microscope in the spirit of OSH and the competition. We invited and encouraged other team members to look at our designs, tinker with them and think about how they might alter them to fit their specific needs.