Difference between revisions of "Part:BBa K4583058"
| Line 24: | Line 24: | ||
* Second, this promoter was placed upstream of the <em> BFP </em> gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts. | * Second, this promoter was placed upstream of the <em> BFP </em> gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts. | ||
For plasmid construction methods and other experimental procedures, see the Design page. | For plasmid construction methods and other experimental procedures, see the Design page. | ||
| − | + | ===Protocols=== | |
| − | === | + | |
Our experimental conditions for characterizing this part were as follows: | Our experimental conditions for characterizing this part were as follows: | ||
* <em>E. coli</em> MG1655 | * <em>E. coli</em> MG1655 | ||
| Line 33: | Line 32: | ||
We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time). | We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time). | ||
| − | === | + | ===Characterization using GFP in 2-plasmids bacteria=== |
| − | In this section we used the PACYC plasmid with <html><a href="https://parts.igem.org/Part: | + | In this section we used the PACYC plasmid with <html><a href="https://parts.igem.org/Part:BBa_K4583003"> BBa_K4583003(PYU16)</a></html> upstream of <em>GFP</em> gene(Fig. 1). We transformed it into L19 and L31 with <html><a href="https://parts.igem.org/Part:BBa_K4583009"> BBa_K4583009(PesaRwt)</a>, <a href="https://parts.igem.org/Part:BBa_K4583010"> BBa_K4583010(PesaRc)</a>, <a href="https://parts.igem.org/Part:BBa_K4583011"> BBa_K4583011(PesaRp)</a></html> plasmids respectively (6 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 2 |
<html> | <html> | ||
<figure> | <figure> | ||
| − | <img src="https://static.igem.wiki/teams/4583/wiki/ | + | <img src="https://static.igem.wiki/teams/4583/wiki/pyu16gfp.png"width="410" height="240"> |
| − | <figcaption><b>Fig. 1 </b>. Genetic Circuit when characterizing | + | <figcaption><b>Fig. 1 </b>. Genetic Circuit when characterizing PYU16 using GFP </figcaption> |
</figure> | </figure> | ||
</html> | </html> | ||
| − | + | Only one combination showed significant differences in expression time and expression intensity (L31-PesaRp-PYU16). | |
| − | + | ||
| − | + | ||
<html> | <html> | ||
<figure> | <figure> | ||
| − | <img src="https://static.igem.wiki/teams/4583/wiki/pesar- | + | <img src="https://static.igem.wiki/teams/4583/wiki/pesar-pyu16-1.png"width="700" height="390"> |
| − | <figcaption><b>Fig. 2 </b>. Characterization results of | + | <figcaption><b>Fig. 2 </b>. Characterization results of PYU16 in the 2-plasmid bacteria</figcaption> |
</figure> | </figure> | ||
</html> | </html> | ||
| − | === | + | ===Characterization using BFP in 3-plasmids bacteria=== |
| − | In this section, we used the PACYC plasmid with PYU7 upstream of the <em>BFP</em> gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with <html> | + | In this section, we used the PACYC plasmid with PYU7 upstream of the <em>BFP</em> gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with <html><a href="https://parts.igem.org/Part:BBa_K4583011"> BBa_K4583011(PesaRp)</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K4583012"> BBa_K4583012(PesaS)</a> plasmid respectively (4 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 4. |
<html> | <html> | ||
<figure> | <figure> | ||
| − | <img src="https://static.igem.wiki/teams/4583/wiki/ | + | <img src="https://static.igem.wiki/teams/4583/wiki/pyu16bfp.png"width="410" height="240"> |
| − | <figcaption><b>Fig. 3 </b>. Genetic Circuit when characterizing | + | <figcaption><b>Fig. 3 </b>. Genetic Circuit when characterizing PYU16 using BFP </figcaption> |
</figure> | </figure> | ||
</html> | </html> | ||
| − | From the characterization results, we can see that there is a significant delay in the expression of this part from the other promoters. | + | From the characterization results, we can see that there is a significant delay in the expression of this part from the other promoters. PYU16 is expressed at the stationary phase and peaks at the late stationary phase (42h). |
We found roughly the same results for both characterizations, but with slightly different onset times. This may be related to the instrumentation used. For this characterization, we used a Molecular Devices SpectraMax i3x, which has a much higher precision. In addition, the difference between the 2-plasmids system and the 3-plasmids system may also account for the difference. | We found roughly the same results for both characterizations, but with slightly different onset times. This may be related to the instrumentation used. For this characterization, we used a Molecular Devices SpectraMax i3x, which has a much higher precision. In addition, the difference between the 2-plasmids system and the 3-plasmids system may also account for the difference. | ||
<html> | <html> | ||
<figure> | <figure> | ||
| − | <img src="https://static.igem.wiki/teams/4583/wiki/pesas- | + | <img src="https://static.igem.wiki/teams/4583/wiki/pesas-pyu16.png"width="300" height="190"> |
| − | <figcaption><b>Fig. 4 </b>. Characterization results of | + | <figcaption><b>Fig. 4 </b>. Characterization results of PYU16 in the 3-plasmids bacteria</figcaption> |
</figure> | </figure> | ||
</html> | </html> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Revision as of 19:11, 11 October 2023
PYU16-RBS(B0034)-E
PYU16-RBS(B0034)-E
Usage
We designed a auto-lysis system based on this part. The auto-lysis system will express at the late stationary phase and peaks at about 40h.
Late Stationary Phase Promoter
When the bacteria enter the stationary phase, the physiological state of the bacteria changes significantly. During this phase, many genes will respond to make timely adjustments. This 4 parts BBa_K4583000 (PYU3), BBa_K4583001 (PYU7), BBa_K4583003 (PYU16), and BBa_K4583004 (PYU92) are the promoters of E. coil. Their most notable feature is that they will express in the late stationary phase. Moreover, they are self-inducible promoters, which means that no additional inducers are needed to be added for expression. Exogenous inducers are expensive and need to be added artificially, whereas self-induced promoters are cost-effective and relatively stable. This part is also very safe because it comes from E. coli MG1655, a commonly engineered bacterium.
- Late stationary phase promoter
- Self-inducible promoter without additional inducers
- Biosafety
Characterization of Late Stationary Phase Promoter PYU7
Our characterization of this part is divided into two main parts.
- First, this promoter was placed upstream of GFP gene, forming a genetic circuit as shown in Fig. 1. This plasmid was transformed into a bacterium containing another plasmid for characterization. Green and red fluorescence were measured at fixed intervals to compare the expression time and intensity of the two.
- Second, this promoter was placed upstream of the BFP gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts.
For plasmid construction methods and other experimental procedures, see the Design page.
Protocols
Our experimental conditions for characterizing this part were as follows:
- E. coli MG1655
- 30oC, 48h, under vigorous shaking
- Plasmid Backbone: PACYC
- Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) and Molecular Devices SpectraMax i3x.
We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).
Characterization using GFP in 2-plasmids bacteria
In this section we used the PACYC plasmid with BBa_K4583003(PYU16) upstream of GFP gene(Fig. 1). We transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) plasmids respectively (6 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 2
Only one combination showed significant differences in expression time and expression intensity (L31-PesaRp-PYU16).
Characterization using BFP in 3-plasmids bacteria
In this section, we used the PACYC plasmid with PYU7 upstream of the BFP gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with BBa_K4583011(PesaRp) and BBa_K4583012(PesaS) plasmid respectively (4 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 4.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]