Team:Cardiff Wales/Improve

With thanks to our sponsors!

Visit our social media pages!

Cardiff iGEM 2018

---

Click to close sidebar ---

---

• Project
  • Description
  • Design
  • Experiments
  • Interlab
  • Plant
• People
  • Team
  • Attributions
• Human Practices
  • Human Practices
  • Public Engagement
  • Bioinformatics
  • Survey
• Collaborations
• Application
  • Modelling
  • Hardware
  • Applied Design
• Parts
  • Parts
  • Basic Parts
  • Composite Parts
  • Part Collection
• Achievements
  • Results
  • Medal Fulfilment
  • Improve
  • Demonstrate
• Lab Book
  • Notebook
  • Protocols
  • Gallery
• Safety

Improve

The part we decided to improve was part BBa_k133055, encoding an mCherry reporter gene. The improved protein is our mCherry, part BBa_K2810009, which has been codon optimised for use in Nicotiana bethamiana and has GoldenGate compatible 5' and 3' ends, containing the BsmBI/Esp3I recognition site, and DNA sequences that allow ligation into the plasmid pSB1C3, forming the BsaI recognition site in the process. This allows parts with compatible sticky ends following BsaI digestion to be ligated into a specific order. Below is a BLASTn result of the two sequences, where INSERT QUERY AND SAMPLE DETAILS HERE.

INSERT ANNOTATED BLAST RESULT HERE

We decided to improve an mCherry part as when using eGFP as a reporter gene we found that it had low expression, and inconsistent results when compared to our GUS reporter. We knew that eGFP functioned as a dimer, requiring more protein expression to get as much equivalent fluorescence as mCherry, which fluoresces as a monomer. eGFP also fluoresces in the green region of visible light. We found that eGFP expression was very similar to background levels, and we suspected this may be due to chlorophyll autofluorescence. However, some sources suggest that chlorophyll produces auto fluorescence in the red region of light, around 680nm, but we found that our mCherry had much clearer, less speculative fluorescence. We found some convincing results and high quality characterisation with our promoters, 35S CaMV and RTBV, when using mCherry as the reporter gene.

Of course, as we were using GoldenGate cloning for our project, we could only use GoldenGate compatible parts to make our composite parts. This means that we were unable to experiment with the original parent part, BBa_k133055, as it is not GoldenGate compatible. For this reason, we only have results for our improved part, which, to our knowledge, is the only GoldenGate compatible mCherry reporter gene in the registry. All the tested constructs that used this improved part are shown below.

INSERT ALL mCHERRY RESULTS HERE

• Codon optimised mCherry for use in plants
• Added Type IIS restriction sites and ligation sites that allow this part to be GoldenGate compatible.

For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.

Gold Medal Criterion #2

Standard Tracks: Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement. Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry. The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.

Special Tracks: Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.