Difference between revisions of "Part:BBa K2442204"

Revision as of 22:47, 1 November 2017

Pmtle + B0032

This part contains the minimal pmtlE promoter and B0032 ribosome binding site. pmtlE is the intergenic region between mtlR and the mtlE coding region in Pseudomonas Flourescens. Naturally the pmtle promoter is regulated by the mtlR protein which complexes upon induction by sugars and activates the promoter.

The intergenic region lies between the mtlR stop codon and the mtlE start codon (158 bp). Within this intergenic region the mtle promoter resides. Based on previous literature we decided to use the minimal sequence needed for promoter activity.

For this part the minimal promoter + B0032. (including the pmtle promoter) was cloned into psb1c3. Within this sequence there is a putative wild type ribosome binding site. This part was one of the variants tested with mtlR to evoke a GFP response via sugar induction.

Usage and Biology

This part was cloned into a plasmid containing GFP. We constructed two plasmid constructs- the regulatory plasmid BBa_K2442202 responsible for the transcription and translation of the MtlR protein, and the reporter plasmid which would allow for a measurable response to the MtlR-xylulose interaction.The reporter plasmid was constructed by first ligating the mtlE promoter into pSB1C3. We ordered the sequence Liu et al (2015) [1] used, which contained the native promoter and RBS. We also decided to experiment with a variety of RBS in order to find the optimal one for our genetic circuit.

Each reporter variant was tested with each sugar and the reporter variants and regulatory plasmid were tested together with each sugar.We tested these combinations with 6 different sugars; mannitol, sorbitol, sucrose, xylose, ribose, and fructose. Our controls were DH5α empty cells, fresh LB, and each plasmid alone. The two constructs were individually tested with a variety of sugars in order to record the fluorescence in the absence of MtlR. Controls were set up without the constructs in order to measure the basal level of GFP fluorescence. Our results showed:

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2442204 short</partinfo>
-This part contains the pmtlE promoter from Pseudomonas Flourescens. The ribosome binding site here has been coupled to the promter , not the entire intergenic region.
+This part contains the minimal pmtlE promoter and B0032 ribosome binding site. pmtlE is the intergenic region between mtlR and the mtlE coding region in <i>Pseudomonas Flourescens</i>. Naturally the pmtle promoter is regulated by the mtlR protein which complexes upon induction by sugars and  activates the promoter.
+The intergenic region lies between the mtlR stop codon and the mtlE start codon (158 bp). Within this intergenic region the mtle promoter resides. Based on previous literature we decided to use the minimal sequence needed for promoter activity.
+For this part the minimal promoter + B0032. (including the pmtle promoter) was cloned into psb1c3. Within this sequence there is a putative wild type ribosome binding site. This part was one of the variants tested with mtlR to evoke a GFP response via sugar induction.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+This part was cloned into a plasmid containing [https://parts.igem.org/Part:BBa_E0040 GFP]. We constructed two plasmid constructs- the regulatory plasmid BBa_K2442202 responsible for the transcription and translation of the MtlR protein, and the reporter plasmid which would allow for a measurable response to the MtlR-xylulose interaction.The reporter plasmid was constructed by first ligating the mtlE promoter into pSB1C3. We ordered the sequence Liu et al (2015) [1] used, which contained the native promoter and RBS. We also decided to experiment with a variety of RBS in order to find the optimal one for our genetic circuit.
+Each reporter variant was tested with each sugar and the reporter variants and regulatory plasmid were tested together with each sugar.We tested these combinations with 6 different sugars; mannitol, sorbitol, sucrose, xylose, ribose, and fructose. Our controls were DH5α empty cells, fresh LB, and each plasmid alone. The two constructs were individually tested with a variety of sugars in order to record the fluorescence in the absence of MtlR. Controls were set up without the constructs in order to measure the basal level of GFP fluorescence. Our results showed:
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