Difference between revisions of "Part:BBa K206000"

 
 
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K206000 short</partinfo>
 
  
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pBAD is an <i>E.coli</i> promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type (<partinfo>I13453</partinfo>) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
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{|
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|<div style="align: center; valign: center; font-family: Arial; font-size: 12pt">
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{|
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|-
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|width="60px"|''Name'':
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|pBAD strong
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|-
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|width="60px"|''Input'':
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|width="100px"|[http://openwetware.org/wiki/Arabinose L-arabinose]
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|-
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|''Output'':
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| PoPS
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|}
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</div>
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<hr width="800px">
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<div class="noprint" style="padding: 10px; color: #ffffff; background-color: #C0C0C0; width: 800px; align: center">
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<center>
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[[Part:BBa_K206000 |Part Main Page]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[Part:BBa_K206000:Design |Part Design]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[Part:BBa_K206000:Characterization |Characterization]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[pBAD Promoter Family |Family]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[Part:BBa_K206000:Experience |Add Data]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</center>
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</div>
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|}
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===Usage and Biology===
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pBAD is an <i>E.coli</i> promoter that is induced by L-arabinose. In the absence of arabinose, the repressor protein AraC (<partinfo>I13458</partinfo>) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription [[Part:BBa_K206000#References|[1]]]. In the presence of arabinose, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription [[Part:BBa_K206000#References|[1]]].
 +
<br>
 +
<br>
 +
K206000 is a variant pBAD promoter with a modified AraI1 site that has been shown both to be responsive to lower concentrations of arabinose and to exhibit a higher maximum expression than the wild type (<partinfo>I13453</partinfo>), as measured by [[Part:BBa_K206002|coupling to a fluorescent reporter]].
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<br>
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<br>
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''What you can do with it:''
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<br>
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At a given level of arabinose input, <partinfo>K206000</partinfo> will provide a higher level of PoPS output than its [[pBAD Promoter Family|family members]], allowing analog device responses. See [http://2009.igem.org/Team:British_Columbia our wiki] for a project that makes use of this property.
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<br>
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<br>
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''Compatibility'':
 +
<br>
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Chassis: Best used in the ''E. coli'' strain [http://cgsc.biology.yale.edu/Strain.php?ID=111773 BW27783], which has been modified to permit homogeneous pBAD promoter expression by substituting the chromosomal arabinose-dependent promoter of AraE (arabinose transporter protein) with a constitutive promoter [[Part:BBa_K206000#References|[2]]].
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<br>
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Backbone: Has been shown to work on plasmid <partinfo>pSB1C3</partinfo>.
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<br>
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Reporter: Has been shown to work with reporters <partinfo>I13507</partinfo> and <partinfo>I763020</partinfo>.
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<br>
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===<span class='h3bb'>Sequence and Features</span>===
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<partinfo>BBa_K206000 SequenceAndFeatures</partinfo>
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<br>
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===References===
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[http://www.ncbi.nlm.nih.gov/pubmed/11102706 [1]] Schlief, R. (2000). Regulation of the L-arabinose operon of ''Escherichia coli''. Trends in Genetics. '''16'''(12):559-565.
 +
<br>
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[http://www.ncbi.nlm.nih.gov/pubmed/11739756 [2]] Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. '''147'''(12):3241-7.
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K206000 parameters</partinfo>
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<!-- -->
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== Characterized by BNU-China 2019 ==
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We characterize pBAD (BBa_K206000) by an induced suicide system, in which pBAD controls the downstream mazF (BBa_K302033) gene that serves as a reporter, which encodes an endoribonuclease that cleaves RNAs at ACA sites and causes the death of microbe [1]. As a result, we can characterize pBAD in a cell density-dependent manner in Escherichia coli K-12.
 +
 
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[[Image:2019 BNU-China BBa K3036005 change.png| border | center | 400px]]<br>
 +
 
 +
In order to characterize pBAD induced by L-arabinose under different concentrations, we take engineered microbe without induction as control group.
 +
 
 +
As is shown in Fig.1, the cell number of experimental groups show a significant decrease, which indicates pBAD can be induced by 1.25μM/L and 2.5μM/L arabinose, and 2.5μM/L can be considered as a more effective concentration.
 +
 
 +
[[Image:2019 BNU-China BBa K302033 change.jpg| border | center | 400px]]<br>
 +
 
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Figure 1 Cell number declines after induction by L-arabinose. It proves that pBAD is induced by L-arabinose.
 +
 
 +
Beyond our project, pBAD is applied for heterologous gene expression due to its advantages, including moderately high expression levels, induction by a low-cost and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly significant to expressing toxins. [2]
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<b>Experimental approach</b>
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1. Transform the plasmids into E. coli DH5α competent cells.
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2. The engineered bacteria are cultured in 200mL LB-ampicillin (50 ng/µl) medium overnight at 37℃, 200rpm;
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3. Equally divide the culture into 90 centrifuge tubes, which is 1mL respectively. Centrifuge them at 4000rpm for 5 minutes. Discard the liquid.
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4. Resuspend 30 tubes of collected bacteria with LB-ampicillin (50 ng/µl) containing 1.25μM/L and 2.5μM/L L-arabinose respectively as experimental groups. Resuspend 30 tubes of bacteria with pure LB-ampicillin (50 ng/µl) medium.
 +
5. Collect 3 tubes of all groups every 6 hours, dilute all of the samples to 107 times and then spread them on solid LB-ampicillin (50 ng/µl) medium separately. At the same time, refresh the medium to maintain the concentration of L-arabinose.
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6. Count the number of colonies in 5 cm2 per plate after cultured for 24 hours at 37℃
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7. Three repicas are tested in each group.
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<b>Reference</b>
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[1] Nigam A, Ziv T, Oron-Gottesman A, Engelberg-Kulka H2019. Stress-induced MazF-mediated proteins in Escherichia coli. mBio 10: e00340-19. doi:10.1128/mBio.00340-19.
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[2] Diana Széliová, Ján Krahulec, Martin Šafránek, et al. Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains[J]. Journal of Biotechnology, 2016, 236:1-9.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K206000 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K654059 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K206000 parameters</partinfo>
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<partinfo>BBa_K654059 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 14:17, 16 October 2019


Name: pBAD strong
Input: [http://openwetware.org/wiki/Arabinose L-arabinose]
Output: PoPS

Part Main Page        Part Design        Characterization        Family        Add Data       

Usage and Biology

pBAD is an E.coli promoter that is induced by L-arabinose. In the absence of arabinose, the repressor protein AraC (BBa_I13458) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription [1]. In the presence of arabinose, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription [1].

K206000 is a variant pBAD promoter with a modified AraI1 site that has been shown both to be responsive to lower concentrations of arabinose and to exhibit a higher maximum expression than the wild type (BBa_I13453), as measured by coupling to a fluorescent reporter.

What you can do with it:
At a given level of arabinose input, BBa_K206000 will provide a higher level of PoPS output than its family members, allowing analog device responses. See [http://2009.igem.org/Team:British_Columbia our wiki] for a project that makes use of this property.

Compatibility:
Chassis: Best used in the E. coli strain [http://cgsc.biology.yale.edu/Strain.php?ID=111773 BW27783], which has been modified to permit homogeneous pBAD promoter expression by substituting the chromosomal arabinose-dependent promoter of AraE (arabinose transporter protein) with a constitutive promoter [2].
Backbone: Has been shown to work on plasmid pSB1C3.
Reporter: Has been shown to work with reporters BBa_I13507 and BBa_I763020.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[http://www.ncbi.nlm.nih.gov/pubmed/11102706 [1]] Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565.
[http://www.ncbi.nlm.nih.gov/pubmed/11739756 [2]] Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. 147(12):3241-7.


Characterized by BNU-China 2019

We characterize pBAD (BBa_K206000) by an induced suicide system, in which pBAD controls the downstream mazF (BBa_K302033) gene that serves as a reporter, which encodes an endoribonuclease that cleaves RNAs at ACA sites and causes the death of microbe [1]. As a result, we can characterize pBAD in a cell density-dependent manner in Escherichia coli K-12.

2019 BNU-China BBa K3036005 change.png

In order to characterize pBAD induced by L-arabinose under different concentrations, we take engineered microbe without induction as control group.

As is shown in Fig.1, the cell number of experimental groups show a significant decrease, which indicates pBAD can be induced by 1.25μM/L and 2.5μM/L arabinose, and 2.5μM/L can be considered as a more effective concentration.

2019 BNU-China BBa K302033 change.jpg

Figure 1 Cell number declines after induction by L-arabinose. It proves that pBAD is induced by L-arabinose.

Beyond our project, pBAD is applied for heterologous gene expression due to its advantages, including moderately high expression levels, induction by a low-cost and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly significant to expressing toxins. [2]

Experimental approach

1. Transform the plasmids into E. coli DH5α competent cells. 2. The engineered bacteria are cultured in 200mL LB-ampicillin (50 ng/µl) medium overnight at 37℃, 200rpm; 3. Equally divide the culture into 90 centrifuge tubes, which is 1mL respectively. Centrifuge them at 4000rpm for 5 minutes. Discard the liquid. 4. Resuspend 30 tubes of collected bacteria with LB-ampicillin (50 ng/µl) containing 1.25μM/L and 2.5μM/L L-arabinose respectively as experimental groups. Resuspend 30 tubes of bacteria with pure LB-ampicillin (50 ng/µl) medium. 5. Collect 3 tubes of all groups every 6 hours, dilute all of the samples to 107 times and then spread them on solid LB-ampicillin (50 ng/µl) medium separately. At the same time, refresh the medium to maintain the concentration of L-arabinose. 6. Count the number of colonies in 5 cm2 per plate after cultured for 24 hours at 37℃ 7. Three repicas are tested in each group.

Reference

[1] Nigam A, Ziv T, Oron-Gottesman A, Engelberg-Kulka H2019. Stress-induced MazF-mediated proteins in Escherichia coli. mBio 10: e00340-19. doi:10.1128/mBio.00340-19. [2] Diana Széliová, Ján Krahulec, Martin Šafránek, et al. Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains[J]. Journal of Biotechnology, 2016, 236:1-9.


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]