Latest revision as of 12:25, 21 October 2019

IMPROVEMENT REFERENCE: Linkoping_Sweden 2019

This part is a modified version of BBa_K2671000 (Group: iGEM18_Linkoping_Sweden), Designed by: Oliver Hild Walett, Johan Larsson

Introduction

pT7-mNG

This is a improved variant of BBa_K2671000. The biobrick codes for mNeonGreen (mNG), which is a fluorescent protein with great intensity. The protein is currently ranked third in intensity, only beaten by mVenus-Q69M (basic) and Skylan-S (photoswitchable).

BBa_K3182300 has a T7-system as well as a 5’-UTR region, instead of the AraC-pBAD system present in the non-improved biobrick BBa_K2671000. By using the T7-RNA-polymerase (T7-RNAP) from the T7 bacteriophage over the native-RNA-polymerase (n-RNAP) in E. coli the expression is greatly increased. This part requires a host carrying the T7-RNAP, such as Escherichia coli BL21 (DE3) which was the chassis we used.

Figure 1. A schematic representation of the improvement. Where the promotor (purple, pBAD) of the previous biobrick (BBa_K2671000) has been exchanged to pT7. Translation enhancing '5-UTR containing g10-L RBS (BBa_K1758100) has been added as well to the improvement. Together with removal of extra bases (named UTR in figure 1) which is non-coding. A BamHI site was added after the His-tag. The blue colors illustrate the protein coding part of the biobricks.

Usage and Biology

Figure 2. A spectral scan of mNeonGreen. Purified mNeonGreen was analyzed with spectrometry to find optimal excitation and emission wavelengths.

Spectral scan

In order to find optimal excitation and emission wavelengths, and to further confirm earlier studies using mNeonGreen, a spectral scan was conducted. The part BBa_K3182300 was inoculated in 100 mL LB-Miller media along with 25 ug/mL chlorampenicol. After reaching an OD₆₀₀ of 0.8, expression of mNeonGreen was initiated with 0.5 mM IPTG. This culture was then left to express mNeonGreen for 16 hours in 30 °C. The cells were then lysed using sonication, 30 % amplitude, 30 seconds on and 30 seconds off. The cell lysate was then centrifuged at 12000 g for 15 minutes and the soluble fraction was saved. This fraction was then loaded onto a Ni-NTA agarose column by Qiagen, and eluted with 250 mM imidazole. The pure mNeonGreen was later used for the spectral scan seen in Figure 2.

Figure 3. Comparison of the old biobrick, BBa_K2671000 (light grey), and the new biobrick BBa_K3182300 (dark grey). Relative fluorescence divided by start OD₆₀₀ after 16 hours is shown. The error bars represent the mean ± SD from four technical replicates.

Expression

To evenly compare the parts, both biobricks were grown to a starting optical density at 600 nm (OD₆₀₀) of 0.4 ± 0.05. Both biobricks were inoculated in LB-Miller media containing 25 ug/mL chloramphenicol. After the cultures reached OD₆₀₀ 0.4, this part was induced using 0.5 mM IPTG and the old part was in induced with 1.5 mM L-arabinose. Aliquotes of the induced cultures were then placed in a 96-well plate and later analyzed with spectrometry. Fluorescence was then measured over 16 hours in 37 °C and excitation wavelength was 505 nm and emission wavelength was 525. The results from this experiment can be seen in Figure 3. As expected the pT7, the improved part, had a fluorescence which was more than twice than BBa_K2671000.