Difference between revisions of "Part:BBa K2559012"

Revision as of 12:50, 15 October 2018

Promoter of RuBisCO(Prbc)

Promoter of ribulose-1,5-bisphosphate carboxylase/oxygenase(RuBisCO)

Usage and Biology

Several promoters have been used in cyanobacteria, such as the Susan S. Golden Laboratory, the promoter PpsbA2 using the photosynthetic system II D1 protein gene psbA2 and the IPTG inducible promoter Ptrc. Among the promoters used in Synechocystis PCC6803 include PpsbA2, a copper ion-inducible promoter PpetE and a nickel ion-inducible promoter, and a bacterial-derived Ptrc and Ptac promoter. The ribulose sulfide 1,5-diphosphate (RuBisCO) is the main soluble protein in cyanobacteria, so its promoter Prbc is considered to be a strong promoter and has been in the cyanobacteria PCC6301, Anabaena PCC7120. And Spirulina PCC7942, Synechocystis sp.pcc6803 successfully applied.[1]

Figure 1 Map of pFQ20[1]

Qi et al., constructed a high-efficiency expression platform pFQ20 with prbc as a promoter, and used tesA protein as a reporter gene to detect the expression of foreign protein in this expression platform.

Figure 2 Map of pFQ25 [1]

Figure 3 Western blotting of testA[1] M: protein marker; 2: the negative control of Syn-LY2（WT）; 3: positive control using a purified his-tagged enzyme;4–5: triplicate samples of strain Syn-FQ25（Transformants）. [1] Luckily，we have received the pFQ20 from Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Science.And,we successfully construct a efficient bacteria cellulose expression platforms in Synechocystis sp. PCC6803.

Figure 4. colony PCR of extracted Synechocystis sp.PCC6803 genome

Transgenic cyanobacteria with BcsZH-ABCD-BglX genes and the cellulose measurement Cyanobacteria glucose content measurement (3 repeats). The same of quality of transferred cyanobacteria with BcsZH-ABCD-BglX gene and wildtype are treated with lysozyme to break out the cell. The glucose represents the content of cellulose. The addition of cellulase can digest the cellulose to glucose then the bacterial cellulose can measured. The distinct differences between the red column and blue column indicate the high expression intensity of bacteria cellulose. Wilcoxon test indicates our data is reliable.

Figure 5. The measurement of cellulose content in Transgenic cyanobacteria which expressed Bcs gene (Bcs Z/H/A/B/C/D and BglX). The smaller is the Pvalue, the higher is the cellulose content.

The parts can further facilitate the in-depth research for other teams!

Reference :

[1].Qi FX, Tan XM, Lü XF. Construction and evaluation of efficient gene expression platforms in Synechocystis sp. PCC6803. Chin J Biotech, 2013, 29(9): 1332?1342. [1]

Sequence and Features