![](https://parts.igem.org/images/partbypart/icon_composite.png)
Part:BBa_K3182300
Contents
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 91
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.
![](/wiki/images/8/82/T--Linkoping_Sweden--mngimprovementsequence.png)
Usage and Biology
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.
![](/wiki/images/4/4f/T--Linkoping_Sweden--mgnen.png)
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.
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