Difference between revisions of "Part:BBa K2762007"

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<partinfo>BBa_K2762007 short</partinfo>
 
<partinfo>BBa_K2762007 short</partinfo>
  
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===Usage and Biology===
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===Background===
 
This composite part is composed of a consecutive promoter BBa_R0010, a strong ribosomal binding site BBa_B0034 and a PRK-encoding gene BBa_K2762003. Phosphoribulokinase (PRK) (EC 2.7.1.19) is an enzyme involved in the Calvin-Benson-Bassham (CBB) cycle that catalyzes the conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-biphosphate (RuBP).
 
This composite part is composed of a consecutive promoter BBa_R0010, a strong ribosomal binding site BBa_B0034 and a PRK-encoding gene BBa_K2762003. Phosphoribulokinase (PRK) (EC 2.7.1.19) is an enzyme involved in the Calvin-Benson-Bassham (CBB) cycle that catalyzes the conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-biphosphate (RuBP).
  
 
==Characterization==
 
==Characterization==
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[[File:T--NCKU Tainan--part BBa K2762007.png|200px|centre]]
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===Expression in E. coli===
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Finally, WC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% separating gel and 4% stacking gel. Proteins were visualized by staining with Coomassie blue R-250 and were scanned with an Image scanner.
 
Finally, WC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% separating gel and 4% stacking gel. Proteins were visualized by staining with Coomassie blue R-250 and were scanned with an Image scanner.
 
The result is shown below.
 
The result is shown below.
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[[File:T--NCKU Tainan--part BBa K2762007 white final .png|500px|centre]]
  
 
===PRK Toxicity Test===
 
===PRK Toxicity Test===
 
RuBP produced from the PRK catalyzed reaction cannot be metabolized by <i>E. coli</i> and the accumulation of RuBP in <i>E. coli</i> would cause cell growth arrest. To examine the relationship between the expression of PRK in <i>E. coli</i> and its growth, we carried out an experiment to measure the growth of <i>E. coli</i> strain DH5α with and without plasmid carrying <i>prk</i> gene, each in separate glucose and xylose M9 mediums after a 12-hour incubation.
 
RuBP produced from the PRK catalyzed reaction cannot be metabolized by <i>E. coli</i> and the accumulation of RuBP in <i>E. coli</i> would cause cell growth arrest. To examine the relationship between the expression of PRK in <i>E. coli</i> and its growth, we carried out an experiment to measure the growth of <i>E. coli</i> strain DH5α with and without plasmid carrying <i>prk</i> gene, each in separate glucose and xylose M9 mediums after a 12-hour incubation.
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We tested PRK in different strains. We first cloned prk into pSB1C3 and transformed into BL21(DE3). After 12 hours, the strain without plasmid could grow up to  1.4 O.D.600 in altered M9 xylose medium. The strain that contains PRK can grew up to 0.75 O.D.600 in normal M9 medium either. In contrast, the PRK strain that grew in altered M9 xylose medium showed no growth at all. The result shows that PRK can suppress/inhibit the growth, which matches to our expectation.
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[[File:T--NCKU_Tainan--Results_Fig_3.PNG|460px|centre]]
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Fig. 3  The result of PRK test in BL21(DE3). The PRK expressing strain is incubated in both normal M9 medium and altered M9 xylose medium to compare with the strain without plasmid. The PRK expressing strain grown in altered M9 xylose showed merely no growth, which proves the function of PRK.
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Although the function of PRK have been confirmed, we would like to lower the expression of it to minimize the growth arrest. We thus cloned the part into pSB3K3, a low copy number plasmid to lower its protein expression. We then compare the growth under high and low copy number plasmid. We found out that pSB3K3 shows a little growth arrest comparing to the strain without plasmid. The growth of it exceed that of PRK expressed in pSB1C3. We can regulate the expression of PRK via high or low copy number plasmid to optimize the growth and carbon fixation efficiency of the bacteria.
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[[File:T--NCKU_Tainan--prk_high_low.jpeg|460px|centre]]
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Fig.4 Compares the growth in M9 xylose medium of PRK expressing strain in high and low copy number plasmid. The low copy number plasmid, pSB3K3, shows a little bit of growth retard compare to non-PRK expressing strain. However, the toxicity is much less than high copy number expressing strain.
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We also transformed pSB3K3-prk into W3110 strain. W3110 is reported to have higher pressure tolerance. The trend of the results is similar to that of the BL21(DE3) but there is no statistically significant between the experiment and the control group. We deduce that PRK can still function in W3110 since the trend matches our expectation. As pSB3K3 is a low copy number plasmid, the expression of protein may be lower than that of high copy number plasmid. The pressure tolerance of W3110 strain may also lessen the toxicity influence by PRK.
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[[File:T--NCKU_Tainan--prkW3110.jpeg|460px|centre]]
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Figure 5. The result of PRK test in W3110
  
 
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<partinfo>BBa_K2762007 parameters</partinfo>
 
<partinfo>BBa_K2762007 parameters</partinfo>
 
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====Reference====
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Fuyu Gong, Guoxia Liu, Xiaoyun Zhai,Jie Zhou, Zhen Cai and Yin Li1 .(2015,Jun 18). Quantitative analysis of an engineered CO<sub>2</sub>-fixing Escherichia coli reveals great potential of heterotrophic CO<sub>2</sub> fixation.<i> Biotechnology for Biofuels.</i>

Latest revision as of 14:11, 17 October 2018


PlacI-B0034-prk-B0015

Background

This composite part is composed of a consecutive promoter BBa_R0010, a strong ribosomal binding site BBa_B0034 and a PRK-encoding gene BBa_K2762003. Phosphoribulokinase (PRK) (EC 2.7.1.19) is an enzyme involved in the Calvin-Benson-Bassham (CBB) cycle that catalyzes the conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-biphosphate (RuBP).

Characterization

Expression in E. coli

SDS-PAGE of PRK

To find out whether the gene prk is successfully expressed in E. coli, we conducted a SDS-PAGE test. The cells were harvested by centrifuging at 10,000×g for 10 min., and then washed with deionized water for 2 times. The cell density was adjusted to an OD600 of 5 as the sample of whole cell (WC, whole cell catalyst). Finally, WC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% separating gel and 4% stacking gel. Proteins were visualized by staining with Coomassie blue R-250 and were scanned with an Image scanner. The result is shown below.

T--NCKU Tainan--part BBa K2762007 white final .png

PRK Toxicity Test

RuBP produced from the PRK catalyzed reaction cannot be metabolized by E. coli and the accumulation of RuBP in E. coli would cause cell growth arrest. To examine the relationship between the expression of PRK in E. coli and its growth, we carried out an experiment to measure the growth of E. coli strain DH5α with and without plasmid carrying prk gene, each in separate glucose and xylose M9 mediums after a 12-hour incubation.


We tested PRK in different strains. We first cloned prk into pSB1C3 and transformed into BL21(DE3). After 12 hours, the strain without plasmid could grow up to 1.4 O.D.600 in altered M9 xylose medium. The strain that contains PRK can grew up to 0.75 O.D.600 in normal M9 medium either. In contrast, the PRK strain that grew in altered M9 xylose medium showed no growth at all. The result shows that PRK can suppress/inhibit the growth, which matches to our expectation.


T--NCKU Tainan--Results Fig 3.PNG

Fig. 3 The result of PRK test in BL21(DE3). The PRK expressing strain is incubated in both normal M9 medium and altered M9 xylose medium to compare with the strain without plasmid. The PRK expressing strain grown in altered M9 xylose showed merely no growth, which proves the function of PRK.


Although the function of PRK have been confirmed, we would like to lower the expression of it to minimize the growth arrest. We thus cloned the part into pSB3K3, a low copy number plasmid to lower its protein expression. We then compare the growth under high and low copy number plasmid. We found out that pSB3K3 shows a little growth arrest comparing to the strain without plasmid. The growth of it exceed that of PRK expressed in pSB1C3. We can regulate the expression of PRK via high or low copy number plasmid to optimize the growth and carbon fixation efficiency of the bacteria.


T--NCKU Tainan--prk high low.jpeg

Fig.4 Compares the growth in M9 xylose medium of PRK expressing strain in high and low copy number plasmid. The low copy number plasmid, pSB3K3, shows a little bit of growth retard compare to non-PRK expressing strain. However, the toxicity is much less than high copy number expressing strain.


We also transformed pSB3K3-prk into W3110 strain. W3110 is reported to have higher pressure tolerance. The trend of the results is similar to that of the BL21(DE3) but there is no statistically significant between the experiment and the control group. We deduce that PRK can still function in W3110 since the trend matches our expectation. As pSB3K3 is a low copy number plasmid, the expression of protein may be lower than that of high copy number plasmid. The pressure tolerance of W3110 strain may also lessen the toxicity influence by PRK.


T--NCKU Tainan--prkW3110.jpeg

Figure 5. The result of PRK test in W3110

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]


Reference

Fuyu Gong, Guoxia Liu, Xiaoyun Zhai,Jie Zhou, Zhen Cai and Yin Li1 .(2015,Jun 18). Quantitative analysis of an engineered CO2-fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation. Biotechnology for Biofuels.