Biosafety

The release of GMO’s in the soil is a sensitive topic. The release into the environment must be as safe as possible to prevent and contain possible danger. To achieve this we provide a system that minimalizes the impact on the soil and controls the spread of genetic material. The system that provides this safety and the low impact consist out of two parts: the Kill switch and a horizontal gene transfer inhibition. These two systems will be linked to each other, in order to induce less metabolic pressure in the designed microorganism and to produce an easy use complete bio safety system.

OVERVIEW

Kill Switch – Overview

This project aims to protect the natural balance within the soil and provide a maximal safety. We engineered a regulatory system that will only activate stable expression of anti-fungals when needed and will self-destruct the bacteria when the bacterium fulfilled its purpose. Whit this system we reassure that there are the least GMO bacteria in the soil as necessary and that they are only active when BananaGuard is needed. The kill switch system will reassure regulated expression by providing a stable expression of the anti-fungal components that activates only during exposure to fusaric acid. After exposure to fusaric acid the kill switch will have a stable expression of toxins that will kill the bacteria. This reassures that the bacterium removes itself when the threat of Fusarium is gone.

To make this we had three main projects :
- The regulative system with promoters from the registry
- A new characterization method that is able to characterize non inducible promoters
- Design, produce and test new promoters that are repressible by multiple repressors

Besides the self-destruction system the kill switch circuit has lots of potential applications. The regulatory properties of this system can be utilized for any genetic system that needs to memorize the input signal and express the output gene when this signal is gone. The input promoter can be changed into the sensing promoter of choice. Likewise, the output gene can be changed giving the system a great flexibility. An even stronger improvement would be the addition intercellular communication, making a collective synchronized memory possible, which enables simple control over vast populations of bacteria equipped with this system.

The Regulatory System

The kill switch regulatory system consist of a two plasmid system as can be seen in figure 1. One will contain the input system, rhamnose promoter regulating the CIλ repressor gene, and the output pcI/Tet regulating the reporter gene. The other one contains a genetic toggle switch based on the principle of Gardner et al 2000 [1].

The toggle switch functions as a memory system for the input signal. In the final system the rhamnose inducible promoter will be exchanged for the fusaric acid induced promoter and the reporter will express a toxin that will kill the cell. We used these repressors because they are proven to work and are well characterized. The promoters we used where the only promoters that could be double repressed by our selection of repressors. The ramose promoter was chosen due to its concentration specific expression and GFP due to its usefulness as reporter gene.
The function of the kill switch can be explained in three states. These states are shown figure 2-4. In the first state only the repressor TetR will be expressed, repressing pcIλ/Tet and pTet. Before this the toggle switch will be set to this state by IPTG inhibiting the binding of LacI to the promoter.

The rhamnose inducible promoter is induced by rhamnose therefore cIλ is expressed, switching the circuit to its second state. In the second state fusaric acid inducible promoter is active upon Fusarium recognition. Therefore cIλ represses pcIλ/Tet and pcIλ/Lac. This leads to a switch within the toggle switch. TetR can no longer repress pTet, therefore LacI is expressed, keeping the toggle switch in its final state, in which TetR is no longer expressed.

When the organism cannot sense rhamnose anymore the third state of the kill switch is induced. In this final state the pcIλ/Tet promoter is not repressed by the cIλ repressor or the TetR repressor leading to the expression GFP. In the final system this GFP will be toxins that will kill the cell.

The in output plasmid is the plasmid with the rhamnose promoter, the CI lambda repressor, CItet promoter and the GFP or toxins . It is assembled with the standard BioBricking method . the end product is assembled into a low copy number plasmid to reduce the metabolic stress, enable faster reactions to input repressors, and lower the GFP production so that it can be measured more accurately. Another low copy plasmid than pSB3K3 can also be used.

Figure 7 shows cells carrying the pSB3K3 plasmid containing the kill switch input output plasmid (BBa_K1493560) that were incubated on LB agar plates containing 0.2% rhamnose and no rhamnose overnight at 37°C.

On an plates without rhamnose the construct should express GFP since the rhamnose promoter is not active so there is no repression of the promoter expressing GFP. On the plates with rhamnose colonies should not express GFP since the rhamnose promoter should be active and producing repressors that repress the promoter that produces GFP. Under UV light the colonies on the plate with no rhamnose shows green fluorescence. The colonies induced by rhamnose don’t show a green colour. From the picture we can conclude that the production of GFP is repressed by the repressor protein cIλ.
Since this plasmid is also used to characterize the PcI/Tet promoter (see rhamnose mediated characterization) it also gives a first suggestion that this method works.

The toggle switch is a memory system consisting out of the PCIlac promoter , TetR gene , Tet promoter and LacI repressor , see figure 8 and 9.

It is assembled with the standard biobricking method . The end product is assembled into a low copy number plasmid. Another low copy plasmid than pSB3K3 can also be used. To test the toggle switch a GFP was added behind the tetR gene.

In the active state, the promoter pTet is on expressing the lac repressor protein LacI, which binds to pcI/lac and inhibits the production of TetR, keeping it in the same state. Besides the lac gene, a second gene is arranged downstream of the tet promoter, the gfp gene. We expect to see a high fluorescence values for the active state, since both LacI and GFP will be expressed. In the resting state only TetR will be produced, repressing pTet, thus no GFP is expressed resulting in low fluorescence. The active state can be induced by adding the chemical anhydrotetracycline (aTc). ATc is an inhibitor of TetR, which induces the toggle switch to start expressing LacI and GFP. The resting state is induced by the chemical Isopropyl β-D-1-thiogalactopyranoside (IPTG), which is an inhibitor of LacI.

To determine if the toggle switch is functional and the repressor proteins produced under the promoters inhibit the gene expression of each other the system was characterized. Three cultures were prepared in M9 medium in duplo, inoculated with Escherichia coli strain NEB5α carrying the toggle switch with reporter. The first containing 500 ng/ml aTc to get the cells into the active state and the second inducing the resting state by 2 mM IPTG as described by Gardner et al[1]. A third culture is grown with no inducer, inducing a random state. E. coli carrying pSB3K3 with cI and lacI with no promoter was used as an auto fluorescence control. Cultures were grown overnight and fluorescence was measured the next morning using a plate reader (395 excitation, 509 emission). Cells did not grow well in M9 with 2mM IPTG, as the OD was low (OD 600 nm of 0.3) compared to OD 600nm 0.6-0.7 of the cultures grown with ATC or with no inducer, after overnight incubation.

Figure 10 shows that the cultures grown in M9 with 500 ng/ml aTc have an average relative fluorescence unit of 7300. The cultures grown with 2 mM IPTG give an average fluorescence unit of 1000. The cells grown in M9 with no inducer give a fluorescence of 5200 RFU. These values indicate that the toggle switch is functional as it has a high fluorescence when grown with aTc, which means it is in its active state. The resting state is reached with low fluorescence when grown with IPTG. We can conclude from the RFU value of the cultures grown with no inducer that the culture has a mix of both states. Thus, the toggle switch is able to choose a random state and no state is significantly more preferred than the other.

The model of the system we build predicted that the system would not be stable with the promoter configurations we had been using so far. These promoters where promoters from the registry with the same operator sites that we needed, but configured in such a way that it was likely they would not work. Therefore promoters with another configuration of the operator sites of the promoter would be necessary. With the results of the model we build 6 new promoters based on the sequences of Elowiz et al 2007 [2].

Promoters have 3 different regions that are separated by the -34 and -10 RNA polymerase binding sites, see figure 12. In the article Elowiz et al 2007 [2] these are called the distal, core and proximal region. Binding sites on these regions have different strengths, generally the core region is the strongest followed by the proximal region and finally the distal region [2] [3].

By putting repressor operator sites on different positions you can influence the strength of the repression [2, 3] and place the most important repressor sites on the strongest regions. In theory systems would be tuneable with this method. Here we give this theory a try by designing promoters for a toggle switch that have operator sites at the same places so that the toggle switch is as balanced as possible. The promoters from the registry where not balanced in this way, so we made new promoters with the sequences provided in the article of Elowiz et al 2007 [2]. In the article they use a set of different sequences for the proximal, core and distal regions that can be combined in different combinations. We used these sequences to design 6 different promoters in silico. The operator configurations of the 6 promoters are listed below:

• Empty site, TetR site, TetR site (-- Tet Tet)
• TetR site, empty site, TetR site (Tet – Tet)
• CIλ site, LacI site, LacI site (CI Lac Lac)
• TetR site, CIλ site, TetR site (Tet CI Tet)
• CIλ site, TetR site, TetR site (CI Tet Tet)
• LacI site, CIλ site, Lac site (Lac CI Lac)

We then ordered two single stranded sequences that had an overlapping region, and used these to make a double stranded promoter whit BioBricking sites. These promoters where ten assembled into pSB1C3 and other combinations to make the kill switch system.
The protocol can be found here