Difference between revisions of "Part:BBa K2442205"

 
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<partinfo>BBa_K2442205 short</partinfo>
 
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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.
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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.
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For this part the minimal promoter + B0034. (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.
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===Usage and Biology===
 
===Usage and Biology===
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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) 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.
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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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[[Image:T-Glasgow-pleaseworkgraph.png|450px|thumb|left|'''Figure 1:''' The top image shows our construct, the part names and their code names. The graph shows OD normalised fluorescence minus L-broth background for each sugar condition. The variant construct plasmids were all tested. The OD reflects the fluorescence of each construct tested in the presence of sugar. Again, ribose and sorbitol show the greatest fluorescence response across the plasmids with the regulatory plus reporter plasmid showing the overall greatest fluorescence levels. The regulatory constructs are in pSB1C3 whilst the reporter plasmids are in PSB3K3. DH5α was used as a control. The bottom table shows the plasmid names and their code names. ]]
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K2442205 SequenceAndFeatures</partinfo>
 
  
  

Revision as of 22:53, 1 November 2017


Pmtle + B0034

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 + B0034. (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) 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:

Figure 1: The top image shows our construct, the part names and their code names. The graph shows OD normalised fluorescence minus L-broth background for each sugar condition. The variant construct plasmids were all tested. The OD reflects the fluorescence of each construct tested in the presence of sugar. Again, ribose and sorbitol show the greatest fluorescence response across the plasmids with the regulatory plus reporter plasmid showing the overall greatest fluorescence levels. The regulatory constructs are in pSB1C3 whilst the reporter plasmids are in PSB3K3. DH5α was used as a control. The bottom table shows the plasmid names and their code names.