Difference between revisions of "Part:BBa K2516001"
Line 37: Line 37:
 
2. Lumbreras, V., Stevens, D. R., & Purton, S. (1998). Efficient foreign gene expression in <i>Chlamydomonas reinhardtii</i> mediated by an endogenous intron. The Plant Journal, 14(4), 441-447.
 
2. Lumbreras, V., Stevens, D. R., & Purton, S. (1998). Efficient foreign gene expression in <i>Chlamydomonas reinhardtii</i> mediated by an endogenous intron. The Plant Journal, 14(4), 441-447.
  
 +
<h1> Characterization by iGEM TU_Kaiserslautern 2019</h1>
 +
<div class="figure">
 +
<img src="https://2019.igem.org/wiki/images/1/1b/T--TU_Kaiserslautern--resultsFigure4.svg"/>
 +
<p class="caption">
 +
<span class="phat">Expression of the enzymes MUT-PETase and MHETase in Chlamydomonas reinhardtii</span>
 +
                                  (a) L2A MoClo construct contain aadA selection cassette, PAR-promoter, HA-tagged MUT-PETase and MHETase, RPL23-terminator. (b) UVM4 cultures transformed with construct L2A were inoculated in TAP at 25°C. Samples of eleven different cultures were taken from shake flasks after 3 days. Lysed cells of each sample were loaded onto the gel and proteins were separated via SDS-PAGE. Proteins were blotted onto a membrane and detected by the anti-HA antibody. Molecular weight marker is indicated as MW. Black arrow represents MHETase, white arrow indicates MUT-PETase. The expression via PAR promoter of both MUT-PETase and MHETase is visible in the colonies 18, 22 and 27. The PAR promoter works efficiently in combination with the enzymes MUT-PETase and MHETase. HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) was used as a positive control.
  
 +
                    </p>
 +
</div>
 +
<p>
 +
In summary, we achieved really high expression levels using the PAR promoter. This might be partially because we used the rubisco introns in our coding sequence. As shown in a paper by schroda, the PAR promoter in combination with rubisco introns can lead to extraordinarily high expression levels<sup>1,2</sup>. To conclude, we can recommend the PAR promoter to anyone working with chlamy. It is the gold standard for a reason and we never felt like we needed to use a different promoter.
 +
</p>
 +
<p>
 +
[1] Schroda, M., Beck, C. F., & Vallon, O. (2002). Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. The Plant Journal, 31(4), 445-455.
 +
</p>
 +
<p>
 +
[2] Lumbreras, V., Stevens, D. R., & Purton, S. (1998). Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. The Plant Journal, 14(4), 441-447.
 +
</p>
 +
</div>
 +
</div>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 03:50, 22 October 2019


pHSP70/pRBCS2 combined promoter for C. reinhardtii


Improved part from 2016

This part is an improved version of USP_UNIFESP-Brazil 2016 iGEM team. Link: BBa_K2136013


The combined constitutive endogenous promoter used for foreign gene expression in C. reinhardtii.

This promoter consists of 5 main parts:

1) HSP70A promoter (1-263) is the first upstream region of Heat Shock Protein 70A from C. reinhardtii. This sequence spacing-dependent and acts as an inhibitor of transcriptional silencing in C. reinhardtii. [1]

2) [Improvement] High-efficiency separator sequence between two promoters (264-272) that provides the best spacing between two promoters, resulting in an increase of transformation rate by 2.6 times relative to original separating sequence. [1]

3) RBCS2 promoter (273-454) is a promoter region of nuclear ribulose bisphosphate carboxylase/oxygenase small subunit gene from C. reinhardtii. It is the most widely utilized promoter for robust transgene expression in C. reinhardtii. [1]

4) 5' untranslated region of the nuclear RBCS2 gene (455-489) from C. reinhardtii acts as an enhancer.

5) [Improvement] The first intron of RBCS2 gene (490-634), it acts as an enhancer and increases expression by 5-6 times [2].


This promoter can be utilized by other iGEM teams as a standardized promoter sequence for constitutive gene expression in C. reinhardtii.


Reference List:

1. Schroda, M., Beck, C. F., & Vallon, O. (2002). Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. The Plant Journal, 31(4), 445-455.

2. Lumbreras, V., Stevens, D. R., & Purton, S. (1998). Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. The Plant Journal, 14(4), 441-447.

Characterization by iGEM TU_Kaiserslautern 2019

<img src="https://2019.igem.org/wiki/images/1/1b/T--TU_Kaiserslautern--resultsFigure4.svg"/>

Expression of the enzymes MUT-PETase and MHETase in Chlamydomonas reinhardtii (a) L2A MoClo construct contain aadA selection cassette, PAR-promoter, HA-tagged MUT-PETase and MHETase, RPL23-terminator. (b) UVM4 cultures transformed with construct L2A were inoculated in TAP at 25°C. Samples of eleven different cultures were taken from shake flasks after 3 days. Lysed cells of each sample were loaded onto the gel and proteins were separated via SDS-PAGE. Proteins were blotted onto a membrane and detected by the anti-HA antibody. Molecular weight marker is indicated as MW. Black arrow represents MHETase, white arrow indicates MUT-PETase. The expression via PAR promoter of both MUT-PETase and MHETase is visible in the colonies 18, 22 and 27. The PAR promoter works efficiently in combination with the enzymes MUT-PETase and MHETase. HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) was used as a positive control.

In summary, we achieved really high expression levels using the PAR promoter. This might be partially because we used the rubisco introns in our coding sequence. As shown in a paper by schroda, the PAR promoter in combination with rubisco introns can lead to extraordinarily high expression levels1,2. To conclude, we can recommend the PAR promoter to anyone working with chlamy. It is the gold standard for a reason and we never felt like we needed to use a different promoter.

[1] Schroda, M., Beck, C. F., & Vallon, O. (2002). Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. The Plant Journal, 31(4), 445-455.

[2] Lumbreras, V., Stevens, D. R., & Purton, S. (1998). Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. The Plant Journal, 14(4), 441-447.

</div> </div>

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 264
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]