Part:BBa_K592000:Experience

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Applications of BBa_K592000

We, UNIST_KOREA (2011 iGEM) team obtained the plasmids encoding the hybrid light receptor, biosynthesis pathway for PCB production and Pompc expressing cI from Christ Voigt.

Characterization of Light receptor and its invertor

Orginally all light regulated promoters repress gene expression in the presence of light. However, for our experiment we need to switch on the expression of the target gene in the presence of light. Hence, we tried to engineer an invertor such that light favors the expression of the target gene. The invertor we exploited was the famous lambda repressor, cI.

In order to avoid the leaky expression of cI in the presence of light and enhance the expression of the target gene, we integrated the Pompc expressing cI into the chromosome of E. coli. We tried to regulate the expression of target gene by light mediated modulation of the level of chromosomally encoded cI (Fig 1).

Effect of duration of light and age of cells on gene expression

We also wanted to characterize the duration of light needed to induce gene expression. Our results indicate that it takes almost 1 hour for the PompC promoter to be inactivated, cI being degraded and the target gene being expressed. The 1 hour duration was irrespective of the stage in which the cells were induced i.e. even when the cells were induced in log phase or in stationary phase, it takes 1 hour for the target gene to be expressed. However, fold change in gene expression differs with the phase in which gene expression was induced (Fig 2).

Figure 2

Cross talk between Cellular function and the Light receptor

The light receptor was a hybrid of the cyanobacterial light receptor and E. coli osmo regulator. Further, the promoter regulated by light was originally regulated by osmolality i.e. higher concentration of salt/sugar would repress the promoter. We have deleted the native EnvZ gene in order to avoid the cellular cross talk. However, despite the deletion of envZ gene, promoter Pompc was regulated by osmolarity (Figure 3). This indicates the presence of a cross-talk between the native cellular function and the light receptor.

Disadvantage The crosstalk observed in the light receptor would be a disadvantageous feature in case light mediated gene expression systems are to be used in industrial process were the osmolarity effects will be high.

2013 SJTU-BioX-Shanghai puts this part into the constant expression operon and uses it as one of our light-controlled gene expression systems to regulate the amount of sgRNA.

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