Difference between revisions of "Part:BBa K3748015"

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(→‎Team Estonia_TUIT 2022 characterization of BBa_K3748015 (pREV1))
 
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==Team Estonia_TUIT characterization of BBa_K3748015 (<i>pREV1</i>)==
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==Team Estonia_TUIT 2022 characterization of BBa_K3748015 (<i>pREV1</i>)==
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<i>pREV1</i> is a constitutive promoter responsible for the expression of <i>REV1</i>, a gene encoding bi-functional DNA-directed DNA polymerase/deoxycytidyl transferase (Lee <i>et al</i>., 2015). This enzyme is involved in error-prone translesion synthesis, one of the pathways for DNA repair (Lawrence, 2004).
  
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<i>pREV1</i> is a constitutive promoter (Michael E. Lee et. al, 2015) responsible for the expression of <i>Rev1</i>, a gene encoding bi-functional  DNA-directed DNA polymerase/deoxycytidyl transferase. This enzyme is involved in error-prone translesion synthesis, one of the pathways for DNA  repair (Lawrence CW, 2004).
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We quantified the expression driven by <i>pREV1</i> 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
  
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<i>pREV1</i> promoter showed constitutive levels of Venus fluorescence throughout the experiment. Compared to the background fluorescence of the DOM90 strain, <i>pREV1</i> demonstrated a five-fold increase in fluorescence intensity, respectively (figure 1). The results confirm the data from Michael E. Lee’s article from 2015, where the <i>pREV1</i> was the weakest promoter present in the kit.
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<i>pREV1</i> promoters showed a constitutive level of Venus fluorescence throughout the experiment, confirming that it is a constitutive promoter. Compared to the background fluorescence of the DOM90 strain <i>pREV1</i> demonstrated a five-fold higher level of fluorescence intensity (Figure 1).
  
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[[Image:PREV1 Estonia TUIT new.png|600px|thumb|center|<b>Figure 1. The expression level of Venus is controlled by pREV1</b>. Bars indicate the mean fluorescence intensity (expressed in arbitrary units, AU) of pREV1-Venus and DOM090 control from the analyzed cell population. Error bars show standard deviation.]]
  
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[[Image:PREV1 Estonia TUIT.png.png|600px|thumb|center|'''Figure 1:''' <b>Graph depicting median fluorescence intensity of <i>pREV1</i> and control strain</b>]]
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Analyzed constitutive promoters can be recommended when constant weak (<i>pREV1</i> promoter) expression of a target gene is required.
  
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References:
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<b>References:</b>
 
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<ul>
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<li>A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. Michael E. Lee, William C. DeLoach, Bernardo Cervantes, and John E. Dueber, 2015</li>   
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<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>   
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<li>Cellular functions of DNA polymerase zeta and Rev1 protein, Lawrence CW (2004), Adv Protein Chem</li>
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<li>Lawrence, C. W. (2004). Cellular functions of DNA polymerase ζ and REV1 protein. <i>Advances in Protein Chemistry, 69, </i>167–203. https://doi.org/10.1016/S0065-3233(04)69006-1</li>
 
</ul>
 
</ul>

Latest revision as of 02:01, 12 October 2022

pREV1

Weak strenght yeast promoter S.cerevisiae.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 705
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Team Estonia_TUIT 2022 characterization of BBa_K3748015 (pREV1)

pREV1 is a constitutive promoter responsible for the expression of REV1, a gene encoding bi-functional DNA-directed DNA polymerase/deoxycytidyl transferase (Lee et al., 2015). This enzyme is involved in error-prone translesion synthesis, one of the pathways for DNA repair (Lawrence, 2004).

We quantified the expression driven by pREV1 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

pREV1 promoters showed a constitutive level of Venus fluorescence throughout the experiment, confirming that it is a constitutive promoter. Compared to the background fluorescence of the DOM90 strain 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 intensity (expressed in arbitrary units, AU) of pREV1-Venus and DOM090 control from the analyzed cell population. Error bars show standard deviation.

Analyzed constitutive promoters can be recommended when constant weak (pREV1 promoter) expression of a target gene is required.

References: