Part:BBa_K3032130

Light-regulated plasmid copy number device intermediate (0.16)

This is an intermediate construct to the light-regulated plasmid copy number system. It consists of an Anderson promoter (BBa_J23116) with light-activated protein EL222 binding sequence inserted between -35 and -10 promoter regions and ColE1 RNA I gene (without promoter).

Introduction

Overview

Light-regulated modules open new ways to precise control of cellular behavior and dramatically accelerates the progress of synthetic biology applications in neuroscience, cardiology, and cell biology. Nevertheless, there are potential issues in a current optogenetic toolbox of prokaryotes as a lack of rapid and switchable control . Transcriptional or translational regulation of gene expression has been the dominant control element used in gene circuits. However, there is still a lack of well-characterized components, such as orthogonal and compatible promoters. As a solution to this problem, the higher-level control module is required.

In 2017 Vilnius-Lithuania iGEM team SynOri established a framework allowing to regulate plasmid copy number in E. coli cells by re-engineered ColE1 origin of replication. ColE1 plasmid replicon based on two antisense RNA molecules: RNA I and RNA II. The transcript of RNA II forms an RNA-DNA duplex with plasmid and acts as a primer for DNA polymerase. For that reason, RNA II is often called a replication initiator. However, another molecule - RNA I may bind to its antisense version of RNA II, which results in replication inhibition. Also, Jayraman et al. (2016) engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light-dependent DNA-binding protein EL222. Inserting EL222 binding sequence between -35 and -10 promoter regions led to a higher than 3-fold reduction in RFP fluorescence when the engineered E. coli was exposed to blue light .

Design

This year we combined RNA I/RNA II with EL222 protein to develop a light-regulated system for plasmid copy number control. For the detection of differences in plasmid copy number, mRFP1 was chosen as a reporter protein.

The design of this system is based on regulating the transcription of RNA I, which is known for inhibiting the replication of plasmids. The idea is to keep plasmid copy number low in the dark state and to increase copy number by exposing cells to blue light. In the dark, RNA I is actively transcribed, which does not allow DNA polymerase to bind to RNA II transcript as RNAI – RNAII duplex is formed. However, the insertion of EL222 binding sequence between -35 and -10 regions of RNA I promoter causes EL222 binding in blue light illumination and inhibited transcription of RNA I. This leads to a reduced formation of RNAII-RNAI duplex and plasmid copy number expansion.

An animation showing the mechanism of post-translational control.

The animation showing the light-regulated plasmid copy number system mechanism of action.

While building our system, we faced several challenges with not being able to predict some required factors easily:

• The amount of EL222, which would be optimal for light-dependent regulation;
• The strength of RNA I promoter that would not over- or under- inhibit replication;
• The strength of mRFP1 promoter, which could be representative of measurements.

Scheme 1 Different variants of promoters used for design of the systems

In order to increase our chances of finding a system with the best functionality, we used three variants of parts for every issue mentioned above. As a result, we obtained 27 variants of systems for light-regulated plasmid copy number, which alter in promoter strength of EL222, RNA I, and mRFP1.

Results

The functionality of these variants was tested in E. coli DH5α cells by growing them in the light or the dark for 6 hours, 37°C. After measurements with a plate reader, the data was estimated by dividing the fluorescence of mRFP1 by OD600. We also used two types of controls:

• Cells without light-regulated plasmid copy number system to test if light has an impact on the growth of bacteria (K);
• The system wherein RNA I promoter with EL222 binding site is replaced to Anderson promoter possessing alternative strength (K1, K4, K7).

Figure 1. Testing variants of the light-regulated plasmid copy number control system after growing bacteria in the dark or light.

According to the obtained data, almost all our light-regulated systems were functional. Both types of control samples revealed no difference between bacteria growing in the dark or the light. The results showed that the best candidates were two samples possessing the highest light/dark ratio of plasmid copy numbers: Sample 17 – EL222 promoter BBa_J23100, RNA I promoter BBa_K3032110, mRFP1 promoter – BBa_J23105. Sample 26 – EL222 promoter BBa_J23110, RNA I promoter BBa_K3032110, mRFP1 promoter – BBa_J23105. These variants were chosen for further experiments to evaluate plasmid copy number dynamics over time. The samples were cultivated in dark or light conditions 37°C for 16 hours. Then the light source was turned off, and cells were cultivated for 8 hours.

Figure 2. Testing the dynamics of the light-regulated plasmid copy number control systems after growing bacteria in the dark or light for 24 hours./

Figure 3. Testing the dynamics of the light-regulated plasmid copy number control systems after growing bacteria in the dark or light for 24 hours./

a) EL222 promoter BBa_J23100, RNA I promoter BBa_K3032110, mRFP1 promoter – BBa_J23105. b) EL222 promoter BBa_J23110, RNA I promoter BBa_K3032110, mRFP1 promoter – BBa_J23105. A dashed line represents the time (16 hour) when the light source was eliminated and all cells were grown in the dark for the remaining experiment] The first thing we noticed in both samples was the growing plasmid copy number light/dark ratio since the 8th hour. After 16 hours plasmid copy number was 1.5-2-fold higher in cells that were exposed to light, which is just a little bit lower than the ratio obtained by Jayraman et al. (2016). Other impressive results obtained after the elimination of light source as plasmid copy number in the light started becoming similar to the one in the dark. According to our results, a light-regulated plasmid copy number system is suitable for dynamic control – one of the key goals in synthetic biology.

References

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 31
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]