Difference between revisions of "Part:BBa K2797003"

Revision as of 16:11, 2 October 2018

mNeonGreen

BBa_K2797003 (mNeonGreen) codon optimised for expression in E. Coli to replace mut3GFP as an improved fluorescent reporter in the interlab study.

Usage and Biology

The mNeonGreen protein is widely used in the imaging of cellular components due to it having a fluorescence 3-5 times that of GFP. Importantly however, it is thought to be more photostable than the mut3GFP used in the InterLab study, although there is little indication in the literature that it has been used as a reporter for the characterisation of circuits. Replacing mut3GFP with mNeonGreen allowed the investigation of whether the fast folding capabilities coupled with its brightness and higher photostability could yield a lower spread of fluorescence values in regard to the original mut3GFP, making it a better tool for part characterisation.

Newcastle 2018 - Interlab Characterisation

Three further InterLab studies were carried out using mNeonGreen expressing E. coli DH5-alpha. These contained the original test devices from the iGEM 2018 distribution kit (Anderson Promoter Collection), using the same conditions as the original study. The fluorescein/OD of the mNeonGreen study was compared to the original InterLab test device data by using a fluorescein calibration curve. Two distinct errors were present in this study:

• TD6 did not successfully transform- Unknown cause, currently thought to be due to failed assembly.
• TD5 reproducibility error - TD5 is actually the positive control. During initial unsuccessful transformations, human error led to TD5 being replaced with the positive control.

Nonetheless, mNeonGreen test devices did

Fluorescence/OD values of the original (white boxes) and mNeonGreen (shaded boxes) test devices at 0 hours and 6 hours post incubation at 37°C @ 220 rpm. A- Colony 1, B- Colony 2.

Sequence and Features