Part:BBa_K2516001

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.

Contribution by iGEM TU Kaiserslautern 2020

The pAR-promotor has, since its introduction to the iGEM-community, been tried and tested. After all its characteristics as a fusion promotor have proven to be efficient in working with Chlamydomonas reinhardtii. It is made made from the promotors from the proteins HSP70 and RBCS2. HSP70 is a heat shock protein, therefore its promotor is slightly heat resistant.¹ While RBCS2 is the gene of the small subunit 2 of RuBisCO and is expressed constitutively.

As such we decided to test the efficiency of the pAR-promotor in combination with the mutated laccase from Botrytis aclada (BaLac) and the laccase from a marine organism (marLac). At first we assembled a Level 2 construct consisting of the spectinomycin-resistance, the coding sequence of the laccases (BaLac or marLac) with the pAR-promotor, a 3xHA-tag for detection and the RPL23 terminator. (as seen in the figure below)


Level 2 construct of cytosolic BaLac (BBa_K3589207) and cytosolic marLac (BBa_K3589208) It includes the constitutive pAR-promotor, the enzyme (BaLac or marLac) fused with a 3xHA tag for detection and a spectinomycin-resistance for selection.


Proof for expression of a) BaLac and b) marLac in Chlamydomonas reinhardtii It shows the Immunoblot of 12 spectinomycin-resistant colonies, which were transformed with a) construct (BBa-K3589207) and b) construct (BBa_K3589208) (as seen in Fig. x). 2 µg Chlorophyll were always loaded onto the gel. At ca. 70 kDA (indicated with an arrow) is the expression of BaLac in the transformants 5,9 and 11 to be seen. At ca. 60 kDA (indicated with an arrow) can the expression of marLac in the transformants 5,9 and 11 be seen. A 3xHA-tagged protein was used as positive control, while the recipient strain (UVM4) was used as a negative control.

As can be seen in the figure above an expression of both BaLac and marLac have been achieved by using the pAR-promotor. Even though activity could not be confirmed, the pAR-promotor still proves to be efficient for expressing in C. reinhardtii and as such can be recommended to everyone working with C. reinhardtii.

References

(1) Schroda, M.; Blöcker, D.; Beck, C. F. The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. The Plant journal : for cell and molecular biology 2000, 21 (2), 121–131. DOI: 10.1046/j.1365-313x.2000.00652.x.​

Characterization by iGEM TU_Kaiserslautern 2019

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 levels^1,2. 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.

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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]