Difference between revisions of "Part:BBa K3748011"
(→‎Team Estonia_TUIT2022 characterization of BBa_K3748011 (pPAB1))
 
(7 intermediate revisions by the same user not shown)
Line 18: Line 18:
 
<!-- -->
 
<!-- -->
  
−
==Team Estonia_TUIT characterization of BBa_K3748015 (<i>pPAB1</i>)==
+
==Team Estonia_TUIT 2022 characterization of BBa_K3748011 (<i>pPAB1</i>)==
  
−
Constitutive <i>pPAB1</i> promoter (Michael E. Lee et. al, 2015), controls a gene encoding the <i>Pab1<i> protein. This protein binds Poly(A) and is involved in the control of poly(A) tail length on nuclear mRNA transcripts (N Amrani et. al, 1997).
+
Constitutive <i>pPAB1</i> (Lee <i>et al</i>., 2015) regulates the expression of an essential gene encoding for poly(A) binding protein, which is involved in the regulation of poly(A) tail length. Pab1 is localized to the nucleus and cytoplasm. It was shown that modulation of Pab1 levels could improve cell fitness under stress conditions (Martani <i>et al</i>., 2015).
  
−
<i>pPAB1</i> promoter showed constitutive levels of Venus fluorescence throughout the experiment. Compared to the background fluorescence of the DOM90 strain, <i>pPAB1</i> showed a 19-fold increase in fluorescence intensity (figure 1).
+
We quantified the expression driven by the <i>PAB1</i> promoter using fluorescent proteins as reporter genes. The expression cassettes were integrated into the yeast genome, and the fluorescence of the reporter proteins was monitored by quantitative time-lapse microscopy. The fluorescence levels were compared to the cell background fluorescence of the control DOM90 strain, which does not express any fluorescent proteins.
  
−
[[Image:PPAB1 Estonia TUIT.png|600px|thumb|center|'''Figure 1:''' <b>Graph depicting median fluorescence intensity of <i>pPAB1</i> and control strain</b>]]
+
<i>pPAB1</i> promoter showed a constitutive level of Venus fluorescence during the experiment, confirming that it is a constitutive promoter. Compared to the background fluorescence of the DOM90 strain, <i>pPAB1</i> showed a 19-fold higher expression level, and <i>pREV1</i> demonstrated a five-fold higher level of fluorescence intensity (figure 1).
 +
 
 +
[[Image:PPAB1 Estonia TUITnew.png|600px|thumb|center|<b>Figure 1. The expression level of Venus is controlled by pREV1</b>. Bars indicate the mean fluorescence signal (AU), and error bars show the standard deviation.]]
 +
 
 +
Analyzed constitutive promoter can be recommended when constant moderate (<i>pPAB1</i> promoter) expression of a target gene is required.
  
 
<b>References:</b>
 
<b>References:</b>
 
<ul>
 
<ul>
−
<li>A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. Michael E. Lee, William C. DeLoach, Bernardo Cervantes, and John E. Dueber, 2015</li>   
+
<li>Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. <i>ACS Synthetic Biology, 4</i>(9), 975–986. https://doi.org/10.1021/SB500366V/SUPPL_FILE/SB500366V_SI_002.ZIP</li>   
−
<li>Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro, N Amrani, M Minet, M Le Gouar, F Lacroute, F Wyers, 1997</li>
+
<li>Martani, F., Marano, F., Bertacchi, S., Porro, D., & Branduardi, P. (2015). The Saccharomyces cerevisiae poly(A) binding protein Pab1 as a target for eliciting stress-tolerant phenotypes. <i>Scientific Reports, 5, </i>18318. https://doi.org/10.1038/SREP18318</li>
 
</ul>
 
</ul>

Latest revision as of 12:13, 11 October 2022


pPAB1

S.cerevisiae medium strenght promoter.

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]


Team Estonia_TUIT 2022 characterization of BBa_K3748011 (pPAB1)

Constitutive pPAB1 (Lee et al., 2015) regulates the expression of an essential gene encoding for poly(A) binding protein, which is involved in the regulation of poly(A) tail length. Pab1 is localized to the nucleus and cytoplasm. It was shown that modulation of Pab1 levels could improve cell fitness under stress conditions (Martani et al., 2015).

We quantified the expression driven by the PAB1 promoter using fluorescent proteins as reporter genes. The expression cassettes were integrated into the yeast genome, and the fluorescence of the reporter proteins was monitored by quantitative time-lapse microscopy. The fluorescence levels were compared to the cell background fluorescence of the control DOM90 strain, which does not express any fluorescent proteins.

pPAB1 promoter showed a constitutive level of Venus fluorescence during the experiment, confirming that it is a constitutive promoter. Compared to the background fluorescence of the DOM90 strain, pPAB1 showed a 19-fold higher expression level, and pREV1 demonstrated a five-fold higher level of fluorescence intensity (figure 1).

Figure 1. The expression level of Venus is controlled by pREV1. Bars indicate the mean fluorescence signal (AU), and error bars show the standard deviation.

Analyzed constitutive promoter can be recommended when constant moderate (pPAB1 promoter) expression of a target gene is required.

References:

  • Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975–986. https://doi.org/10.1021/SB500366V/SUPPL_FILE/SB500366V_SI_002.ZIP
  • Martani, F., Marano, F., Bertacchi, S., Porro, D., & Branduardi, P. (2015). The Saccharomyces cerevisiae poly(A) binding protein Pab1 as a target for eliciting stress-tolerant phenotypes. Scientific Reports, 5, 18318. https://doi.org/10.1038/SREP18318