Difference between revisions of "Part:BBa K3182300"
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<partinfo>BBa_K3182300 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3182300 SequenceAndFeatures</partinfo> | ||
<br> | <br> | ||
+ | |||
+ | ==IMPROVEMENT REFERENCE: Linkoping_Sweden 2019== | ||
+ | This part is a modified version of <partinfo>BBa_K2671000</partinfo> (Group: iGEM18_Linkoping_Sweden), Designed by: Oliver Hild Walett, Johan Larsson | ||
<h1>Introduction</h1> | <h1>Introduction</h1> | ||
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<partinfo>BBa_K3182300 short</partinfo> | <partinfo>BBa_K3182300 short</partinfo> | ||
− | 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). | + | This is a improved variant of <partinfo>BBa_K2671000</partinfo>. 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). |
− | + | ||
<br><br> | <br><br> | ||
− | + | <partinfo>BBa_K3182300</partinfo> has a T7-system as well as a 5’-UTR region, instead of the AraC-pBAD system present in the non-improved biobrick <partinfo>BBa_K2671000</partinfo>. 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 <i>Escherichia coli</i> BL21 (DE3) which was the chassis we used. | |
− | + | [[File:T--Linkoping_Sweden--mngimprovementsequence.png|900px|thumb|left|<b>Figure 1.</b> A schematic representation of the improvement. Where the promotor (purple, pBAD) of the previous biobrick (<partinfo>BBa_K2671000</partinfo>) has been exchanged to pT7. Translation enhancing '5-UTR containing g10-L RBS (<partinfo>BBa_K1758100</partinfo>) 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.]] | |
− | [[File:T--Linkoping_Sweden--mngimprovementsequence.png|900px|thumb|left|<b>Figure 1.</b> 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.]] | + | |
<br><br><br><br><br> | <br><br><br><br><br> | ||
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<br><br><br> | <br><br><br> | ||
<br><br><br> | <br><br><br> | ||
− | + | <br><br><br> | |
+ | <h1>Usage and Biology</h1> | ||
+ | [[File:T--Linkoping_Sweden--mngspectra.png|350px|thumb|left|<b>Figure 2.</b> A spectral scan of mNeonGreen. Purified mNeonGreen was analyzed with spectrometry to find optimal excitation and emission wavelengths.]] | ||
− | < | + | <h2>Spectral scan</h2> |
− | [[File:T--Linkoping_Sweden--mgnen.png| | + | In order to find optimal excitation and emission wavelengths, and to further confirm earlier studies using mNeonGreen, a spectral scan was conducted. The part <partinfo>BBa_K3182300</partinfo> was inoculated in 100 mL LB-Miller media along with 25 ug/mL chlorampenicol. After reaching an OD<sub>600</sub> 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. |
+ | |||
+ | <br><br><br> | ||
+ | <br> | ||
+ | |||
+ | [[File:T--Linkoping_Sweden--mgnen.png|350px|thumb|left|<b>Figure 3.</b> Comparison of the old biobrick, <partinfo>BBa_K2671000</partinfo> (light grey), and the new biobrick <partinfo>BBa_K3182300</partinfo> (dark grey). Relative fluorescence divided by start OD<sub>600</sub> after 16 hours is shown. The error bars represent the mean ± SD from four technical replicates.]] | ||
+ | |||
+ | <h2>Expression</h2> | ||
+ | To evenly compare the parts, both biobricks were grown to a starting optical density at 600 nm (OD<sub>600</sub>) of 0.4 ± 0.05. Both biobricks were inoculated in LB-Miller media containing 25 ug/mL chloramphenicol. After the cultures reached OD<sub>600</sub> 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 <partinfo>BBa_K2671000</partinfo>. | ||
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Latest revision as of 12:25, 21 October 2019
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.