Part:BBa_K2797007

Test Device 4 for the iGEM InterLab Study (mNeonGreen)

BBa_K2797007, test device 4 (originally BBa_J364007 for the iGEM 2018 InterLab study with mNeonGreen replacing the GFP fluorescent reporter. Each mNeonGreen test device retains the same promoter, RBS and terminator of each of their corresponding InterLab test device vectors, with the only change being mNeonGreen replacing the GFPmut3b.

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 standard curve.

Figure 1 shows the fluorescein/OD values of the original InterLab devices (GFPmut3b) vs the mNeonGreen test devices. The test device 4 promoter, J23100, is the strongest promoter in the InterLab study and has the highesy fluorescein/OD of all test devices in the original InterLab study. However the original test device 4 has the largest spread of data throughout the test devices. The mNeonGreen variant of test device 4 reduces the spread of data significantly while also increasing the fluorescein/OD values. This points towards mNeonGreen being a useful alternative to GFPmut3b in the InterLab study and part characterisation.

Fluorescein/OD values of the original (white boxes) and mNeonGreen (shaded boxes) test devices at 6 hours post incubation at 37°C @ 220 rpm for 2 separate colonies. Fluorescein/OD is shown on the y-axis, while the test devices and controls of both the original and mNeonGreen devices are shown on the x-axis. At hour 0, throughout the GFP and mNeonGreen devices there are large spreads of data. At hour 6, mNeonGreen devices can be seen to have a smaller spread of data when compared to GFP in both colonies 1 & 2. Also, in most cases, the fluorescence is much higher in the mNeonGreen, with mNeonGreen TD4 having the highest median fluorescein/OD value (1.57) of the whole study. The pattern of fluorescence regarding the most productive devices is the same as that of the original InterLab study. Each test device group had 4 replicates carried out with the error bars showing the standard deviation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 39
  
• 1000
  COMPATIBLE WITH RFC[1000]