Difference between revisions of "Part:BBa K4729001"
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<partinfo>BBa_K4729001 short</partinfo>
 
<partinfo>BBa_K4729001 short</partinfo>
 
__NOTOC__
 
__NOTOC__
 +
 +
==Background==
 +
 +
 +
Most bacteria prefer sulfate as their sulfur source, yet its availability is generally restricted in natural environments. When faced with sulfate limitation Sinorhizobium meliloti and numerous other bacteria produce sulfate starvation-induced proteins, including ABC-type transporters crucial for taurine (2-aminoethanesulfonate) uptake, which can be used as an alternative sulfur source. The GntR-like transcription factor binds to DNA via helix-turn-helix motif and activates the transcription of the tauAp promoter in the presence of taurine (Wiethaus et al., 2008). Taurine is an advantageous inducer, because it is permeable to the cell membrane and inexpensive, additionany, it has been shown to have tightly regulated expression in S. meliloti (Mostafavi et al., 2014), as such, we believe it to be a valuable resource for future iGEM teams.
 +
 +
==Characterization & Measurement==
 +
 +
Measurements of expression strength were done using luminescence measurements
 +
and the LUX operon. Luminescence measurement are significantly more sensitive
 +
in comparison to fluorescence, allowing the characterization of weaker
 +
regulatory elements.The strains used in measurement
 +
experiments were grown in MOPS minimal medium liquid cultures containing the
 +
appropriate antibiotic. The use of minimal medium reduces possible variations
 +
due to inconsistencies in rich medium components. In order to
 +
maximize homogeneity between sample, the optical density of liquid cultures was measured
 +
before each measurement and diluted down to the same value using lab automation
 +
Platforms.
 +
 +
<html>
 +
  <center>
 +
  <figure>
 +
    <img src='https://static.igem.wiki/teams/4729/wiki/results/composite-with-different-promoters.png' width='800px' />
 +
    <figcaption>
 +
        <p>
 +
          <b>
 +
          Figure 1: Characterization construct for basic parts in <i>A. rhizogenes</i></b> <br>
 +
          This construct was designed for portability between <i>E. coli</i> and Alphaproteobacteria and compatibility with the MoClo Golden Gate standard as a level 1 entry vector (Döhlemann et al., 2017; Weber et al., 2011)  <br>
 +
                </figcaption>
 +
  </figure>
 +
  </center>
 +
</html>
 +
 +
==Results==
 +
 +
 +
<html>
 +
  <center>
 +
  <figure>
 +
    <img src='https://static.igem.wiki/teams/4729/wiki/results/comparing-all-inducible-promoters.png' width='800px'
 +
alt='Measurement and comparison of different inducible promoter systems in A. rhizogenes' />
 +
    <figcaption>
 +
        <p>
 +
          <b>
 +
          Figure 2: </b> Testing different inducible promoter systems in A. rhizogenes ARqua1.
 +
    </figcaption>
 +
  </figure>
 +
  </center>
 +
</html>
 +
 +
 +
 +
The results presented in figure 2 show the luminescence produced by induced vs not induced cultures in the mid-log phase. Out of the eleven promoters tested, only 4 displayed a significant difference in expression level between induced and not induced cultures: Ptac, Pvan, Pnahr and Ptau. Pbad and Pcym also had some response to the induction, albeit extremely faint. From those four promoters, we observe that Ptac and Pvan had the highest overall basal expression, and Ptau the lowest. In addition, Ptau did display the lowest leakiness of all systems tested. Overall, PnahR appeared to have a good middle ground between expression strength and expression tightness. However, upon further investigation, it was found that growth of  A. rhizogenes ARqua1 cultures in liquid medium was inhibited by  sodium salicylate (Figure 2). In Agrobacterium tumefacies C58, PnttgR, Ptet and Pcym have been shown to have a stronger response than the one observed by our experiment, additionally the toxicity of sodium salicylate was not observed (Qian et al., 2021; Schuster & Reisch, 2021).
 +
 +
 +
  
 
taken from:
 
taken from:
Line 9: Line 65:
 
Seiler, V. N. (2023). Improved inducible switches for the implementation of genetic tools in Sinorhizobium meliloti and Ensifer adhaerens (Master's thesis, Philipps-University Marburg)
 
Seiler, V. N. (2023). Improved inducible switches for the implementation of genetic tools in Sinorhizobium meliloti and Ensifer adhaerens (Master's thesis, Philipps-University Marburg)
 
<br><br>
 
<br><br>
 +
Mostafavi, M., Lewis, J. C., Saini, T., Bustamante, J. A., Gao, I. T., Tran, T. T., King, S. N., Huang, Z., & Chen, J. C. (2014). Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression. BMC Microbiology, 14, 295. https://doi.org/10.1186/s12866-014-0295-2
 +
<br><br>
 +
Wiethaus, J., Schubert, B., Pfänder, Y., Narberhaus, F., & Masepohl, B. (2008). The GntR-like regulator TauR activates expression of taurine utilization genes in Rhodobacter capsulatus. Journal of Bacteriology, 190(2), 487–493. https://doi.org/10.1128/JB.01510-07
 +
 +
 +
Döhlemann, J., Wagner, M., Happel, C., Carrillo, M., Sobetzko, P., Erb, T. J., Thanbichler, M., & Becker, A. (2017). A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti. ACS Synthetic Biology, 6(6), 968–984. https://doi.org/10.1021/acssynbio.6b00320
 +
 +
 +
Qian, Y., Kong, W., & Lu, T. (2021). Precise and reliable control of gene expression in Agrobacterium tumefaciens. Biotechnology and Bioengineering, 118(10), 3962–3972. https://doi.org/10.1002/bit.27872
 +
 +
 +
Schuster, L. A., & Reisch, C. R. (2021). A plasmid toolbox for controlled gene expression across the Proteobacteria. Nucleic Acids Research, 49(12), 7189–7202. https://doi.org/10.1093/nar/gkab496
 +
 +
 +
Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. https://doi.org/10.1371/journal.pone.0016765
  
−
<html>
 
−
  <center>
 
−
      <figure>
 
−
        <img src='https://static.igem.wiki/teams/4729/wiki/results/comparing-all-inducible-promoters.png' width='800px'
 
−
alt='Measurement and comparison of different inducible promoter systems in A. rhizogenes' />
 
−
        <figcaption>
 
−
            <p>
 
−
              <b>
 
−
              Figure 1: </b> Testing different inducible promoter systems in A. rhizogenes ARqua1.
 
−
        </figcaption>
 
−
      </figure>
 
−
  </center>
 
−
</html>
 
−
Ptau inducible promoter, induced by taurine.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 13:04, 12 October 2023

Ptau - inducible promoter system


Background

Most bacteria prefer sulfate as their sulfur source, yet its availability is generally restricted in natural environments. When faced with sulfate limitation Sinorhizobium meliloti and numerous other bacteria produce sulfate starvation-induced proteins, including ABC-type transporters crucial for taurine (2-aminoethanesulfonate) uptake, which can be used as an alternative sulfur source. The GntR-like transcription factor binds to DNA via helix-turn-helix motif and activates the transcription of the tauAp promoter in the presence of taurine (Wiethaus et al., 2008). Taurine is an advantageous inducer, because it is permeable to the cell membrane and inexpensive, additionany, it has been shown to have tightly regulated expression in S. meliloti (Mostafavi et al., 2014), as such, we believe it to be a valuable resource for future iGEM teams.

Characterization & Measurement

Measurements of expression strength were done using luminescence measurements and the LUX operon. Luminescence measurement are significantly more sensitive in comparison to fluorescence, allowing the characterization of weaker regulatory elements.The strains used in measurement experiments were grown in MOPS minimal medium liquid cultures containing the appropriate antibiotic. The use of minimal medium reduces possible variations due to inconsistencies in rich medium components. In order to maximize homogeneity between sample, the optical density of liquid cultures was measured before each measurement and diluted down to the same value using lab automation Platforms.

Figure 1: Characterization construct for basic parts in A. rhizogenes
This construct was designed for portability between E. coli and Alphaproteobacteria and compatibility with the MoClo Golden Gate standard as a level 1 entry vector (Döhlemann et al., 2017; Weber et al., 2011)

Results

Measurement and comparison of different inducible promoter systems in A. rhizogenes

Figure 2: Testing different inducible promoter systems in A. rhizogenes ARqua1.


The results presented in figure 2 show the luminescence produced by induced vs not induced cultures in the mid-log phase. Out of the eleven promoters tested, only 4 displayed a significant difference in expression level between induced and not induced cultures: Ptac, Pvan, Pnahr and Ptau. Pbad and Pcym also had some response to the induction, albeit extremely faint. From those four promoters, we observe that Ptac and Pvan had the highest overall basal expression, and Ptau the lowest. In addition, Ptau did display the lowest leakiness of all systems tested. Overall, PnahR appeared to have a good middle ground between expression strength and expression tightness. However, upon further investigation, it was found that growth of A. rhizogenes ARqua1 cultures in liquid medium was inhibited by sodium salicylate (Figure 2). In Agrobacterium tumefacies C58, PnttgR, Ptet and Pcym have been shown to have a stronger response than the one observed by our experiment, additionally the toxicity of sodium salicylate was not observed (Qian et al., 2021; Schuster & Reisch, 2021).



taken from:

Mostafavi, M., Lewis, J. C., Saini, T., Bustamante, J. A., Gao, I. T., Tran, T. T., King, S. N., Huang, Z., & Chen, J. C. (2014). Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression. In BMC Microbiology (Vol. 14, Issue 1). Springer Science and Business Media LLC. https://doi.org/10.1186/s12866-014-0295-2

and

Seiler, V. N. (2023). Improved inducible switches for the implementation of genetic tools in Sinorhizobium meliloti and Ensifer adhaerens (Master's thesis, Philipps-University Marburg)

Mostafavi, M., Lewis, J. C., Saini, T., Bustamante, J. A., Gao, I. T., Tran, T. T., King, S. N., Huang, Z., & Chen, J. C. (2014). Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression. BMC Microbiology, 14, 295. https://doi.org/10.1186/s12866-014-0295-2

Wiethaus, J., Schubert, B., Pfänder, Y., Narberhaus, F., & Masepohl, B. (2008). The GntR-like regulator TauR activates expression of taurine utilization genes in Rhodobacter capsulatus. Journal of Bacteriology, 190(2), 487–493. https://doi.org/10.1128/JB.01510-07


Döhlemann, J., Wagner, M., Happel, C., Carrillo, M., Sobetzko, P., Erb, T. J., Thanbichler, M., & Becker, A. (2017). A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti. ACS Synthetic Biology, 6(6), 968–984. https://doi.org/10.1021/acssynbio.6b00320


Qian, Y., Kong, W., & Lu, T. (2021). Precise and reliable control of gene expression in Agrobacterium tumefaciens. Biotechnology and Bioengineering, 118(10), 3962–3972. https://doi.org/10.1002/bit.27872


Schuster, L. A., & Reisch, C. R. (2021). A plasmid toolbox for controlled gene expression across the Proteobacteria. Nucleic Acids Research, 49(12), 7189–7202. https://doi.org/10.1093/nar/gkab496


Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. https://doi.org/10.1371/journal.pone.0016765


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 182
    Illegal PstI site found at 1411
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1411
    Illegal NotI site found at 602
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 610
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 182
    Illegal PstI site found at 1411
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 182
    Illegal PstI site found at 1411
    Illegal NgoMIV site found at 431
    Illegal NgoMIV site found at 1219
  • 1000
    COMPATIBLE WITH RFC[1000]