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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{|
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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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|}
 
===Usage and Biology===
 
===Usage and Biology===
−
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 (see Figure 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 (Figure 1).
+
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]].
 +
<br>
 +
<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>
 +
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]]].
 +
<br>
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Backbone: Has been shown to work on plasmid <partinfo>pSB1C3</partinfo>.
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<br>
 +
Reporter: Has been shown to work with reporters <partinfo>I13507</partinfo> and <partinfo>I763020</partinfo>.
  
−
[[Image:BritishColumbia-AraC.jpg‎|center|200px|thumb|Figure 1. AraC induction by L-arabinose. [http://www.ncbi.nlm.nih.gov/pubmed/8980677 Reference.]]]
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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>
 +
[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.
  
−
K206000 is a pBAD promoter with a variant AraI1 site that has been shown to be responsive to lower concentrations of arabinose than the wild type (<partinfo>I13453</partinfo>); additionally, it exhibits a higher maximum of protein expression, as measured by coupling to a fluorescent reporter.
+
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
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<partinfo>BBa_K206000 parameters</partinfo>
 +
<!-- -->
  
−
<p>
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== 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.
 +
 +
[[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>
 +
 +
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.
 +
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℃
 +
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.
 +
[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
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===Usage and Biology===
 +
 +
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
−
<partinfo>BBa_K206000 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K654059 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
−
<partinfo>BBa_K206000 parameters</partinfo>
+
<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]