Difference between revisions of "Part:BBa K3171172"

Latest revision as of 21:58, 21 October 2019

Vibrio natriegens Native P1 promotor with mCherry

V. natriegens has been reported to have a remarkable doubling time of 9.8 min. As is the case for the better-studied but slower-growing E. coli, V. natriegens increases its number of ribosomes with the growth rate in order to achieve its extraordinarily high rate of protein synthesis. Multiple mechanisms contribute to this high ribosome synthesis efficiency, including high rRNA gene copy number, strong promoters that contain near-consensus −10, −35, and UP elements and activation by the transcription factor Fis. In addition, V. natriegens rRNA promoters exhibit the relatively short-lived open-complex characteristic of rRNA promoters in E. coli, potentially contributing to the regulation of these promoters in vivo. The native promoter P1 is a constitutive promoter. We fused the the native P1 promoter with mCherry fluorescent protein with the help of the 3A assembly cloning method.

The P1 promoter Part:BBa_K3171171 with Biobrick prefix and suffix was cloned with mCherry to obtain a composite part that constitutively expresses mCherry Part:BBa_J06602. The P1-mCherry plasmid was cloned into a backbone plasmid of Chloramphenicol resistance (pSB1C3).

The gene under the control of this promoter is transcribed constitutively. Fluorescence measurements of the construct in E. coli and V. natriegens reveal a constitutive expression of the reporter mCherry for both organisms. Interestingly our measurements show that the expression levels in E. coli are a lot higher than in V. natriegens as the fold change is 225 for E. coli compared to 3 for V. natriegens (figure 1).

Figure 1: Fluorescence of the reporter mCherry under control of the constitutive P1 promoter in V. natriegens and E. coli in comparison to control without plasmid.

Usage and Biology

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal prefix found in sequence at 7
Illegal suffix found in sequence at 305
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 7
Illegal SpeI site found at 306
Illegal PstI site found at 320
Illegal NotI site found at 13
Illegal NotI site found at 313
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 7
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found in sequence at 7
Illegal suffix found in sequence at 306
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found in sequence at 7
Illegal XbaI site found at 22
Illegal SpeI site found at 306
Illegal PstI site found at 320
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3171172 short</partinfo>
-V. natriegens has been reported to have a remarkable doubling time of 9.8 min. As is the case for the better-studied but slower-growing E. coli, V. natriegens increases its number of ribosomes with the growth rate in order to achieve its extraordinarily high rate of protein synthesis. Multiple mechanisms contribute to this high ribosome synthesis efficiency, including high rRNA gene copy number; strong promoters that contain near-consensus −10, −35, and UP elements; and activation by the transcription factor Fis. In addition, V. natriegens rRNA promoters exhibit the relatively short-lived open-complex characteristic of rRNA promoters in E. coli, potentially contributing to the regulation of these promoters in vivo. The native promoter P1 is a constitutive promoter. The native promoter P1 was fused with mCherry fusion protein with the help of 3A assembly cloning method.
+<i>V. natriegens</i> has been reported to have a remarkable doubling time of 9.8 min. As is the case for the better-studied but slower-growing <i>E. coli</i>, <i>V. natriegens</i> increases its number of ribosomes with the growth rate in order to achieve its extraordinarily high rate of protein synthesis. Multiple mechanisms contribute to this high ribosome synthesis efficiency, including high rRNA gene copy number, strong promoters that contain near-consensus −10, −35, and UP elements and activation by the transcription factor Fis. In addition, <i>V. natriegens</i> rRNA promoters exhibit the relatively short-lived open-complex characteristic of rRNA promoters in <i>E. coli</i>, potentially contributing to the regulation of these promoters in vivo. The native promoter P1 is a constitutive promoter. We fused the the native P1 promoter with mCherry fluorescent protein with the help of the 3A assembly cloning method.
+The P1 promoter [[Part:BBa_K3171171]] with Biobrick prefix and suffix was cloned with mCherry to obtain a composite part that constitutively expresses mCherry [[Part:BBa_J06602]].
+The P1-mCherry plasmid was cloned into a backbone plasmid of Chloramphenicol resistance (pSB1C3).
+The gene under the control of this promoter is transcribed constitutively. Fluorescence measurements of the construct in <i>E. coli</i> and <i>V. natriegens</i> reveal a constitutive expression of the reporter mCherry for both organisms. Interestingly our measurements show that the expression levels in E. coli are a lot higher than in <i>V. natriegens</i> as the fold change is 225 for <i>E. coli</i> compared to 3 for <i>V. natriegens</i> (figure 1).
+[[File:Screenshot 2019-10-21 at 11.47.50.png|400px|]]
+Figure 1: Fluorescence of the reporter mCherry under control of the constitutive P1 promoter in <i>V. natriegens</i> and <i>E. coli</i> in comparison to control without plasmid.
 ===Usage and Biology===
-The native promoter P1 was fused with mCherry fusion protein with the help of 3A assembly cloning method. The P1 promoter with Biobrick prefix and suffix was cloned with mCherry to obtain a composite part that constitutively expressed mCherry (BBa_J06504)
-The P1-mCherry plasmid was cloned into a backbone plasmid of Chloramphenicol resistance (pSB1C3)
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