Difference between revisions of "Part:BBa K1152009:Experience"

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For validation and characterization of sfp as functional PPtase we used the indigoidine producing NRPS indC from ''P. luminiscense''. This one module NRPS is able to convert glutamine into the bright blue pigment indigoidine.
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For validation and characterization of sfp as functional PPtase we used the indigoidine producing NRPS indC from ''P. luminescence''. This one module NRPS is able to convert glutamine into the bright blue pigment indigoidine.
 
We used this part in a pSB3K3-derived plasmid with a lac promoter (<partinfo>BBa_R0010</partinfo>) and the RBS <partinfo>BBa_B0029</partinfo> for coexpression with pSB1C3 derived plasmids with following engineered indC deivatives:
 
We used this part in a pSB3K3-derived plasmid with a lac promoter (<partinfo>BBa_R0010</partinfo>) and the RBS <partinfo>BBa_B0029</partinfo> for coexpression with pSB1C3 derived plasmids with following engineered indC deivatives:
 
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With indC as measuring device it is possible to determine effiency of phosphopantetheinyl transferase reaction of sfp. The following figures  show indigoidine production derived from OD measurements. Because indigoidine has its absorbance maxima around 600 nm we had to take an additional OD value into account. To summarize measurements at OD800 and OD590 are used for calculation of indigoidine production. Detailed methodology is described at the iGEM 2013 Heidelberg project website ([http://2013.igem.org/Team:Heidelberg/Indigoidine|Heidelberg 2013 - Indigoidine]).
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Detecting the amount of the NRP expressed by the bacterial host strain is desirable e.g. in order to determine the efficiency of phosphopantetheinyl transferase reaction of sfp. By tagging the NRP with indigoidine, the amount of the fusion peptide can be determined by quantifying the amount of blue pigment present in the cells. Quantification of the pure indigoidine pigment can be easily achieved by optical density (OD) measurements at its maximum wavelength of about 590 nm.
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<gallery widths="400px" heights="300px">
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[[File:IndPD_Fig5.png|280px|thumb|Figure 1: applied from Myers 2012]]
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File:Heidelberg2013_spectra_pRB23-T8+pRB15.png|File 1: Absorbtion spectra measurements over time for an indC construct (pRB23_T8) which does not produce indigoidine
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File:Heidelberg2013_spectra_pRB23-T12+pRB15.png|File 2: Absorbtion spectra measurements over time for an indC construct (pRB23_T12) which does produce indigoidine
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File:Heidelberg2013_ind_pRB23-T8+pRB15.png|File 3: Derived indigoidine production for an indC construct with T8 domain
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File:Heidelberg2013_ind_pRB23-T12+pRB15.png|File 4: Derived indigoidine production for an indC construct with T12 domain
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</gallery>
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===Applications of BBa_K1152009===
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In cellular culture, indigoidine quantification by OD measurements is impaired. Cellular density of liquid cultures is standardly measured as the optical density (OD) at a wave length of 600 nm, i. e. the absorption peak of indigoidine interferes with the measurement of cell density at the preferred wave length (compare to Figure 1, grey dashed line). Thus, for measurement of NRP expression without time consuming a priori purification of the tagged-protein, a method to separate the cellular and pigment-derived contributions to the OD is required (compare to Figure 1, brown and blue lines, respectively). The method of choice, as described by Myers et al.[2013], requires the OD measurement of cell culture at two distinct wavelengths: the robust wave length ODR and the sensitive wave length ODS. The concentration of indigoidine will have to be deducted from measurements at OD800 (ODR) and OD590 (ODS).
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Detailed methodology is described at the iGEM 2013 Heidelberg project website ([http://2013.igem.org/Team:Heidelberg/Project/Indigoidine Heidelberg 2013 - Indigoidine]).
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[[File:Heidelberg2013_indigoidine_production.png|280px|thumb|'''Figure 2:''' Absorbtion spectra measurements over time for two indC constructs (left: pRB23_T10; right: pRB23_T12) with various PPtase helper constructs.]]
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===Applications of BBa_K1152010===
 
This part can be used in order to manufacture NRPS activating constructs. (Examples can be seen at our project site:  
 
This part can be used in order to manufacture NRPS activating constructs. (Examples can be seen at our project site:  
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[http://2013.igem.org/Team:Heidelberg/Indigoidine|Heidelberg 2013 - Indigoidine])
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[http://2013.igem.org/Team:Heidelberg/Project/Indigoidine Heidelberg 2013 - Indigoidine])
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===References===
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* Myers, J. a, Curtis, B. S., & Curtis, W. R. (2013). Improving accuracy of cell and chromophore concentration measurements using optical density. BMC Biophysics, 6(1), 4. doi:10.1186/2046-1682-6-4
  
 
===User Reviews===
 
===User Reviews===

Revision as of 17:39, 6 October 2013


For validation and characterization of sfp as functional PPtase we used the indigoidine producing NRPS indC from P. luminescence. This one module NRPS is able to convert glutamine into the bright blue pigment indigoidine. We used this part in a pSB3K3-derived plasmid with a lac promoter (BBa_R0010) and the RBS BBa_B0029 for coexpression with pSB1C3 derived plasmids with following engineered indC deivatives:

indC derivative T domain
BBa_K1152015 T8
BBa_K1152016 T10
BBa_K1152017 T12
BBa_K1152018 T13
BBa_K1152019 T14

Detecting the amount of the NRP expressed by the bacterial host strain is desirable e.g. in order to determine the efficiency of phosphopantetheinyl transferase reaction of sfp. By tagging the NRP with indigoidine, the amount of the fusion peptide can be determined by quantifying the amount of blue pigment present in the cells. Quantification of the pure indigoidine pigment can be easily achieved by optical density (OD) measurements at its maximum wavelength of about 590 nm.

Figure 1: applied from Myers 2012

In cellular culture, indigoidine quantification by OD measurements is impaired. Cellular density of liquid cultures is standardly measured as the optical density (OD) at a wave length of 600 nm, i. e. the absorption peak of indigoidine interferes with the measurement of cell density at the preferred wave length (compare to Figure 1, grey dashed line). Thus, for measurement of NRP expression without time consuming a priori purification of the tagged-protein, a method to separate the cellular and pigment-derived contributions to the OD is required (compare to Figure 1, brown and blue lines, respectively). The method of choice, as described by Myers et al.[2013], requires the OD measurement of cell culture at two distinct wavelengths: the robust wave length ODR and the sensitive wave length ODS. The concentration of indigoidine will have to be deducted from measurements at OD800 (ODR) and OD590 (ODS).

Detailed methodology is described at the iGEM 2013 Heidelberg project website ([http://2013.igem.org/Team:Heidelberg/Project/Indigoidine Heidelberg 2013 - Indigoidine]).

Figure 2: Absorbtion spectra measurements over time for two indC constructs (left: pRB23_T10; right: pRB23_T12) with various PPtase helper constructs.

Applications of BBa_K1152010

This part can be used in order to manufacture NRPS activating constructs. (Examples can be seen at our project site: [http://2013.igem.org/Team:Heidelberg/Project/Indigoidine Heidelberg 2013 - Indigoidine])

References

  • Myers, J. a, Curtis, B. S., & Curtis, W. R. (2013). Improving accuracy of cell and chromophore concentration measurements using optical density. BMC Biophysics, 6(1), 4. doi:10.1186/2046-1682-6-4

User Reviews

UNIQ396389f19c165f81-partinfo-00000007-QINU UNIQ396389f19c165f81-partinfo-00000008-QINU