Chemical regulation of Fea1 driven transgene expression in Chlamydomonas reinhardtii
Chemical regulation of Fea1 driven transgene expression in Chlamydomonas reinhardtii
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DOI：
10.1016/j.algal.2017.08.006
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发表时间：
2017-09-01
影响因子：
5.1
通讯作者：
Stute, Stephanie Christine
中科院分区：
文献类型：
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作者：
Coutinho, Paula Barjona do Nascimento;Friedl, Christine;Stute, Stephanie Christine
诱导型启动子可以提供调节的基因表达，允许在最合适的培养时刻生物合成基因产物。在该研究中，铁响应性Fea 1启动子的诱导和失活的参数（艾伦et al.，2007）在绿色衣藻莱茵衣藻中进行了研究。我们的研究结果表明，使用的构建体，ble-2A-mCherry，可以成功地表达Fea 1启动子在缺铁条件下。通过流式细胞术获得的荧光蛋白mCherry的荧光信号可在不同强度下检测到，以响应培养基中0 μ M至20 μ M范围内的铁浓度。我们还证明，铁螯合剂deferroxamine（DFO）添加到铁充满介质导致启动子激活，导致mCherry荧光的增加。启动子诱导的可逆性已经在将细胞转移到无铁螯合剂的培养基后3小时内检测到。在这种情况下，mCherry荧光的逐渐降低可以在48小时内达到低至在用DFO处理40小时中观察到的荧光的5%。DFO处理后的细胞活力不受高达100 μ M螯合剂浓度的影响，这使得能够建立诱导和阻遏的循环过程，以在Fea 1诱导型启动子的控制下产生重组蛋白。
Inducible promoters can provide regulated gene expression allowing the biosynthesis of gene products at most suitable moments of cultivation. In this study, parameters of induction and deactivation of the iron-responsive Fea1 promoter (Allen et al., 2007) were investigated in the green alga Chlamydomonas reinhardtii. Our results indicate that the construct used, ble-2A-mCherry, can be expressed successfully by the Fea1 promoter under iron-deficient conditions. The fluorescence signals of the fluorescent protein mCherry obtained via flow cytometry were detectable at different intensities in response to concentrations of iron ranging from 0 mu M to 20 mu M in media. We also demonstrate that the addition of the iron chelator deferroxamine (DFO) to iron-replete media leads to promoter activation, resulting in the increase of mCherry fluorescence. Reversibility of promoter induction is detected already within 3 h after transferring the cells to iron-replete chelator-free media. In this case, the progressive decrease in mCherry fluorescence can reach, within 48 h, as low as 5% of the fluorescence observed in a 40 h -treatment with DFO. Cell viability after DFO treatment is not affected up to a concentration of 100 mu M of the chelator, which enables the establishment of a cyclic process of induction and repression for the production of recombinant proteins under the control of the Fea1 inducible promoter.