Part:BBa_K3927003

EL222 Expression Driver

This composite part encodes for a 3C120-CYC-LacO promoter (BBa K3927002 ) controlling EL222 for a feed forward circuit, with Part BBa_K2407003 as a terminator

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 670
  Illegal XbaI site found at 203
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 670
  Illegal NheI site found at 185
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 670
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 670
  Illegal XbaI site found at 203
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 670
  Illegal XbaI site found at 203
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

The EL222 expression driver is a composite part consisting of parts BBa_K3927002(3C120-CYC-LacO), BBa_K3570021(NLS-VP16-EL222) and BBa_K2407003(CYCt). 3C120-CYC-LacO is activated by dimerization of NLS-VP16-EL222 in the presence of blue light, which further increases the amount of NLS-VP16-EL222 produced by this part. The amount of NLS-VP16-EL222 then increases in a positive feedback loop, activating both the 3C120-CYC-LacO within this part as well as any other parallel, C120 motif-containing promoters until blue light is removed and NLS-VP16-EL222 un-dimerizes.

Usage

EL222 expression driver can be used to increase and sustain expression of C120 dependent promoters for longer periods of time in S.cerevisiae. This also decouples NLS-VP16-EL222 expression from the cell’s native metabolism by providing a self-sustaining loop that doesn’t rely on native trans-activating elements.

Design

In order for EL222 expression driver’s feedforward loop to kickstart, NLS-VP16-EL222 has to be first expressed from a separate transcriptional unit to drive initial expression of this part. LacO element in 3C120-CYC-LacO also has to be de-repressed, thus LacI must either be absent or disabled by a secondary input, allowing for greater control over this increase in expression.

Characterization

EL222 expression driver was genome integrated into BY4741 S.cerevisiae, alongside NLS-VP16-EL222 expressed from the native ACT1 promoter to kickstart the positive loop. Efficiency of the part was measued by transforming cells with an episomal plasmid containing the 3C120-CYC-LacO promoter upstream of the reporter gene mKO. Fluorescence after culturing in the dark or light for 6 hours was compared to BY4741 harboring the reporter plasmid and only the starter ACT1p-NLS-VP16-EL222 cassette and BY4741 harbouring the reporter plasmid only (Figure 1). To test the ability of the part to sustain expression, the experiment was repeated with induction over 24 hours (Figure 2).

Figure 1: Effect of the EL222 expression driver on reporter gene mKO under the control of 3C120-CYC-LacO after 6 hours of expression, compared to NLS-VP16-EL222 expressed under a native yeast promoter and yeast expressing no NLS-VP16-EL222.

Figure 2: Effect of the EL222 expression driver on reporter gene mKO under the control of 3C120-CYC-LacO after 24 hours of expression, compared to NLS-VP16-EL222 expressed under a native yeast promoter and yeast expressing no NLS-VP16-EL222.

It was demonstrated that the driver circuit was able to increase expression of C120-based 3C120-CYC-LacO, and sustain that increased expression over 24 hours.

Significance

Increasing promoter activity, especially of synthetic promoters, is of great interest in synthetic biology. Common methods include increasing the number of cis-activating elements as well as optimizing the TATA box and translation start sites. Aside from providing the framework for more active optogenetic promoters, we hope that the forward positive feedback loop used here provides a novel method to improve and sustain expression in other systems.