Difference between revisions of "Part:BBa K2997008"
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<partinfo>BBa_K2997008 short</partinfo>
 
<partinfo>BBa_K2997008 short</partinfo>
  
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BBa_K2997008 hello
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===Background===
  
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pchR
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For our device, we decided to make a sensor for sensing p-Cresol. According to our research, we have discovered a group of bacteria known as Pseudomonas can sense p-Cresol naturally. In a previous study, the capability of Pseudomonas to sense p-Cresol is commonly used in the detection of p-cresol in the environment.  
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Achievements:
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    Construction of pchR-GFP BBa_K2997008 and confirm by double digestion.
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    The pchR-GFP (BBa_K2997008) was constructed from IDT DNA synthesis in the form of 3 gBlock gene fragments via overhang PCR. Then, we performed digestion on the finished part and ligated it into the pSB1C3 vector. Subsequently, we transformed the pchR-GFP construct (BBa_K2997008) into E. coli Top 10 using electroporation. After allowing the transformed E. coli Top 10 to grow on LB with Cm (Chloramphenicol) plates, colony PCR and sequencing was carried out to determine successful clones. The colony PCR results indicated that the E. coli has pSB1C3 with pchR-GFP. However, after several attempts to construct pchR-GFP, all the sequencing resulted in mutations.  
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===Part Construction & Experimental Results===
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The pchR-GFP (BBa_K2997008) was constructed from IDT DNA synthesis in the form of 3 gBlock gene fragments via overhang PCR. Then, we performed digestion on the finished part and ligated it into the pSB1C3 vector. Subsequently, we transformed the pchR-GFP construct (BBa_K2997008) into <i>E. coli</i> Top 10 using electroporation. After allowing the transformed <i>E. coli</i> Top 10 to grow on LB with Cm (Chloramphenicol) plates, colony PCR and sequencing was carried out to determine successful clones. The colony PCR results indicated that the <i>E. coli</i> has pSB1C3 with pchR-GFP. However, after several attempts to construct pchR-GFP, all the sequencing resulted in mutations.  
  
 
<<Insert pchR sequencing result2.png below the above paragraphs>>
 
<<Insert pchR sequencing result2.png below the above paragraphs>>
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Fig. 15. Sequencing results of the pSB1C3 w/ pchR-GFP construct.
 
Fig. 15. Sequencing results of the pSB1C3 w/ pchR-GFP construct.
  
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    These results led us to hypothesize that the pchR protein is toxic to our E. coli. Moreover, according to a research paper [9] we have found, it is suggested in the study that p-Cresol is toxic to Gram-negative bacteria, which includes E. coli. Hence, this plasmid construct has been dropped.
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These results led us to hypothesize that the pchR protein is toxic to our <i>E. coli</i>. Moreover, according to a research paper [9] we have found, it is suggested in the study that p-Cresol is toxic to Gram-negative bacteria, which includes <i>E. coli</i>. Hence, this plasmid construct has been dropped.
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    Therefore, the construction of pchR-GFP was carried out again by re-doing overlap PCR, where we combine all the three IDT gene fragments into the full length pchR-GFP construct. For this time, the finished full length pchR-GFP part was then inserted into the vector pBBR1 MCS-4 (carries ampicillin resistance gene).
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<<Insert https://2019.igem.org/wiki/images/a/af/T--NCKU_Tainan--pCHR_double_digest_2.jpg
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below the above paragraphs>>
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Therefore, the construction of pchR-GFP was carried out again by re-doing overlap PCR, where we combine all the three IDT gene fragments into the full length pchR-GFP construct. For this time, the finished full length pchR-GFP part was then inserted into the vector pBBR1 MCS-4 (carries ampicillin resistance gene).
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<img src="https://2019.igem.org/wiki/images/a/af/T--NCKU_Tainan--pCHR_double_digest_2.jpg" style="width:30%;">
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Fig. 16. Gel electrophoresis result of pBBR1 MCS-4 with full length pchR-GFP. Lane 1: Marker; Lane 2: Pre-ligation of vector pBBR1 MCS-4 (4950bp) and insert pchR-GFP (3138bp); Lane 3: Post-ligation of pBBR1 MCS-4 with full pchR-GFP (8098 bp)  
 
Fig. 16. Gel electrophoresis result of pBBR1 MCS-4 with full length pchR-GFP. Lane 1: Marker; Lane 2: Pre-ligation of vector pBBR1 MCS-4 (4950bp) and insert pchR-GFP (3138bp); Lane 3: Post-ligation of pBBR1 MCS-4 with full pchR-GFP (8098 bp)  
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After that, we proceeded into transforming the construct into Pseudomonas fluorescens 55 and Pseudomonas aeruginosa PAD1 via electroporation.  
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After that, we proceeded into transforming the construct into <i>Pseudomonas fluorescens</i> 55 and <i>Pseudomonas aeruginosa</i> PAD1 via electroporation.  
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However, due to the complications surrounding the selection marker being used (Ampicillin), we were not able to get a definitive result that indicates that we have positive colonies. This is due to the fact that P.fluorescens 55 and P.aeruginosa PAD1 are naturally resistant to ampicillin and the vector we used for our construct carries ampicillin resistance gene. Hence, positive colonies cannot be differentiated with negative colonies. Besides that, our colony PCR results on the single colonies were also proven to be negative.  
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However, due to the complications surrounding the selection marker being used (Ampicillin), we were not able to get a definitive result that indicates that we have positive colonies. This is due to the fact that <i>P.fluorescens</i> 55 and <i>P.aeruginosa</i> PAD1 are naturally resistant to ampicillin and the vector we used for our construct carries ampicillin resistance gene. Hence, positive colonies cannot be differentiated with negative colonies. Besides that, our colony PCR results on the single colonies were also proven to be negative.  
  
 
Therefore, with time being one of the factors, we decided to drop any further construction and functional experiments surrounding the p-Cresol sensing part (pchR-GFP).   
 
Therefore, with time being one of the factors, we decided to drop any further construction and functional experiments surrounding the p-Cresol sensing part (pchR-GFP).   
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
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Our construct is primarily centered upon Pseudomonas fluorescens (PC24), because it has a gene cluster that consists of a positive inducible operon, pchR, which allows it to sense p-Cresol and promote the transcription of the downstream region to degrade the p-Cresol. PchR is the gene that translates into activator proteins that can bind with the inducer (p-Cresol) and leads to the conformation of the protein. As a result, the activator protein binds with p-Cresol and the eventual protein-p-Cresol complex will then bind onto the p-Cresol sensing region that is placed just downstream of pchR and start transcription of the gene downstream.
  
 
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Revision as of 13:15, 19 October 2019


Native promoter-pchR-sfGFP

Background

For our device, we decided to make a sensor for sensing p-Cresol. According to our research, we have discovered a group of bacteria known as Pseudomonas can sense p-Cresol naturally. In a previous study, the capability of Pseudomonas to sense p-Cresol is commonly used in the detection of p-cresol in the environment.


Part Construction & Experimental Results

The pchR-GFP (BBa_K2997008) was constructed from IDT DNA synthesis in the form of 3 gBlock gene fragments via overhang PCR. Then, we performed digestion on the finished part and ligated it into the pSB1C3 vector. Subsequently, we transformed the pchR-GFP construct (BBa_K2997008) into E. coli Top 10 using electroporation. After allowing the transformed E. coli Top 10 to grow on LB with Cm (Chloramphenicol) plates, colony PCR and sequencing was carried out to determine successful clones. The colony PCR results indicated that the E. coli has pSB1C3 with pchR-GFP. However, after several attempts to construct pchR-GFP, all the sequencing resulted in mutations.

<<Insert pchR sequencing result2.png below the above paragraphs>>

Fig. 15. Sequencing results of the pSB1C3 w/ pchR-GFP construct.

These results led us to hypothesize that the pchR protein is toxic to our E. coli. Moreover, according to a research paper [9] we have found, it is suggested in the study that p-Cresol is toxic to Gram-negative bacteria, which includes E. coli. Hence, this plasmid construct has been dropped.

Therefore, the construction of pchR-GFP was carried out again by re-doing overlap PCR, where we combine all the three IDT gene fragments into the full length pchR-GFP construct. For this time, the finished full length pchR-GFP part was then inserted into the vector pBBR1 MCS-4 (carries ampicillin resistance gene).


Fig. 16. Gel electrophoresis result of pBBR1 MCS-4 with full length pchR-GFP. Lane 1: Marker; Lane 2: Pre-ligation of vector pBBR1 MCS-4 (4950bp) and insert pchR-GFP (3138bp); Lane 3: Post-ligation of pBBR1 MCS-4 with full pchR-GFP (8098 bp) After that, we proceeded into transforming the construct into Pseudomonas fluorescens 55 and Pseudomonas aeruginosa PAD1 via electroporation.


However, due to the complications surrounding the selection marker being used (Ampicillin), we were not able to get a definitive result that indicates that we have positive colonies. This is due to the fact that P.fluorescens 55 and P.aeruginosa PAD1 are naturally resistant to ampicillin and the vector we used for our construct carries ampicillin resistance gene. Hence, positive colonies cannot be differentiated with negative colonies. Besides that, our colony PCR results on the single colonies were also proven to be negative. Therefore, with time being one of the factors, we decided to drop any further construction and functional experiments surrounding the p-Cresol sensing part (pchR-GFP).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 3173
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal NheI site found at 1769
    Illegal SpeI site found at 3174
    Illegal PstI site found at 3188
    Illegal NotI site found at 7
    Illegal NotI site found at 3181
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
    Illegal BamHI site found at 1182
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 3174
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 3174
    Illegal PstI site found at 3188
    Illegal NgoMIV site found at 1227
    Illegal AgeI site found at 210
    Illegal AgeI site found at 1110
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1136
    Illegal BsaI.rc site found at 3096