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Results and Discussions

Introduction and background

Overall introduction into designed kill switch:

We designed and built a kill switch mechanism that is based on the activation of a lethal gene downstream of an inducible promoter that is activated over time.

Genetically modified organisms, once released into nature, influence the natural diversity of the environment and raise concerns among the public. To reduce this risk, we strive to develop a time-controlled kill switch. By introducing a genetic circuit into E.coli we aim to tightly regulate the bacteria's lifetime according to initial conditions set by the user.

Our kill switch depends on critical acyl homoserine lactone (AHL) concentrations as constitutive expression of inactive LuxR (transcription factor; active form LuxR-AHL complex ) and AiiA (degrades AHL) leads to expression of TetR (repressor protein) repressing ccdB expression (toxic gene) while the AHL concentration decreases over time. If AHL concentrations reach a threshold lower than 1 nM (based on our results), TetR expression stops and Ccdb is expressed causing the cell to die.

Figure 1: Genetic circuit

Background

AHLs (1), (N-acylhomoserine lactones), known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many gram-negative bacteria. The conservation and potent activity of these signaling molecules is based on the lactone ring (see Figure 2).

Figure 2: General chemical structure of an N-acyl homoserine lactone

AiiA (1,2), is an enzyme that degrades N-acyl homoserine lactones. It comes from the bacteria Bacillus sp. 240B1. This AHL-lactonase catalyzes the following reaction hence inactivating the AHLs:

Figure 3: AiiA, an AHL lactonase, catalyzes the hydrolysis of N-acylhomoserines, hence inactivating the potent lactone ring of AHL

LuxR (3),is a protein that can bind to AHL and form an active transcription factor. This LuxR-AHL complex binds to its cognate binding sites of the lux promoter (pLux) and stimulate transcription. The well studied operon of the pLux promoter is originally isolated from the bacterium Vibrio fischeri. The following scheme shows the Lux operon mechanism (Figure 4).

Figure 4: schematic representation for the action of the Lux operon (3).

TetR (4,5), is a repressor protein that inactivates the promotor tet (ptet) which is constitutively active, disabling transcription after being bound by the TetR protein.The tet operon derives from the bacteria Escherichia coli.

Ccdb (6,7), is a toxic protein, that interferes with the activity of DNA gyrase by forming a covalent GyrA-DNA complex that cannot be resolved, thus promoting breakage of plasmid and chromosomal DNA. CcdB comes from the ccd system on the Escherichia coli F plasmid, where it acts as a gyrase poison.

Characterization of first module

Design of BioBrick

In order to understand each module of our system, we started to test and characterize the first part of the genetic circuit. To do so, we built a BioBrick consisting of a constitutive promoter (BBa_I14032) driving expression of LuxR followed by a Lux promoter (BBa_R0062). The Lux promoter is inactive in absence of the AHL-LuxR complex and can be activated when AHL is present. Downstream of the pLux promoter we fused a gene encoding a florescent protein, in this case yellow fluorescent protein (YFP) (see Figure 1A).

Figure 1A : BBa_1767010

This BioBrick (BBa_1767010) was used as a device that allows to

(1) determine the functionality of the promoter pLux.

(2) the formation of the active transcription factor LuxR in presence of AHL.

(3) compare to a similar BioBrick that contains AiiA (BBa_K1767009).

The BioBrick that does contain AiiA (LINK!!!), an AHL-inactivating enzyme, is expressed constitutively upstream of LuxR and degrades AHL hence the amount of AHL available for the formation of the active AHL-LuxR complex is limited. Decreasing active AHL-LuxR complexed leads to a reduced activation of the pLux promoter that should be observed in a reduced activation of YFP over time (Figure 1B).

Figure 1B : BBa_1767009

Experimental Setup

In order to test and compare these BioBricks an overnight starter culture of E.coli harboring the plasmids BBa_K1767009 or BBa_K1767010, respectively were diluted in a low-growth, low-autofluorescence buffer (Bioassay buffer, BA; for 1l we added: 0.5 g Tryptone 0.3 ml Glycerol, 5.8 g NaCl, 50 ml 1M MgSo4, 1ml – 10 x PBS and filled it up with 950 ml DDW). 3-oxohexanoyl-homoserine lactone (3OC6-HSL, Sigma Aldrich (#K3007), herewith referred to as AHL) was added ranging from concentrations of 0.1 to 100000 nM and fluorescence measurements were taken every 30 minutes starting 120 min post-induction over range of 8-12 hours.

Results

As shown in Figure 2, fluorescence values of each timepoint were normalized by dividing fluorescence intensities by OD, averaged over a period of 90 min and 360 min, respectively, and plotted as a function of increasing inducer/AHL concentration.

Figure 2B

Figure 2A

Figure 2A shows the averaged, normalized fluorescence values for the first 2-4 hours (green line with markers) and 6-12 hours (blue line with markers) for the construct without AiiA (BBa_K1767010), while Figure 2B represents the construct harboring AiiA (BBa_K1767009). As can be seen in Figure 2A, the pLux promoter responds to AHL in a dose-dependent manner after a lag-time of 2-4 hours post-induction starting at concentrations of approximately 1 nM of AHL reaching a plateau at higher concentrations of AHL (10000 nM). This observation is similar to previous results shown in the iGEM community (see Figure 3 and BBa_K228010)

Figure 3

Discussions

(1)This comes into play when looking at Figure 2B. Here, we see similar averaged, normalized fluorescence values for both time frames (2-4 hours, green line with markers and 6-12 hours, blue line with markers), howerver no clear induction of YFP expression in absence of AHL is observed, and no significant difference is seen in presence (0 nM AHL) and higher concentrations (10000 nM) compared to a clear, dose-response curve for the construct lacking AiiA (Figure 2A).

(2)What lacks in the characterization of the pLux promoter of previous iGEM teams is the time component that we analyzed during our set of experiments. There is a lag-phase of the pLux promoter being activated as LuxR needs to be expressed, than, after forming the active AHL-LuxR complex avtivate the pLux promoter which in turn expressed YFP which was measured in our experimental setup.

Given the lack of inducible behavior for the AiiA-containing BioBrick (Figure 2B, BBa_1767001), we assume that AiiA, the AHL-degrading enzyme, degrades AHL in an effective and fast manner hence no AHL remains after the start of the fluorescence measurements of approximately 120 min.

1. We can use different RBSs for controlling the expression of Aiia.

2. Use different environmental conditions based on the knowledge we have on Aiia activity depending on variations of pH, temp., etc.

3. Use a different type of Aiia.

4. Use a different type of AHL - different diffusion constant, different reaction behaviour with Aiia, different self-degregation constant etc.

5. Put Aiia under a different type of promotor - not a constant type, but one that is similar to the pLUX - A lot of AHL -> more production of Aiia, and vice versa.

Characterization of genetic circuit

Design of BioBrick

This is the full construct of our genetic circuit that consists of a constitutive promoter (BBa_I14032) driving expression of AiiA followed LuxR and by the Lux promoter (BBa_R0062). The Lux promoter is inactive in absence of the AHL-LuxR complex and can be activated when AHL is present driving expression of TetR followed by a Tet promoter (R0040). The Tet promoter is inactive in presence of the TetR complex and can be activated when TetR is absent. Downstream of the TetR promoter we fused a gene encoding a fluorescent protein, in this case red fluorescent protein (RFP) (see Figure 1C). As proof-of-principle, ccdB the toxic effector gene, was replaced with RFP as gene expression of a fluorescent protein can be studied easily.

Figure 1C : BBa_1767003

This BioBrick (BBa_1767003) contains AiiA, an AHL-inactivating enzyme, that is expressed constitutively downstream of LuxR and degrades AHL hence the amount of AHL available for the formation of the active AHL-LuxR complex is limited. Decreasing the active AHL-LuxR complex leads to a reduced activation of the pLux promoter that results in a reduced activation of TetR gene expression over time. Decreasing the amount of produced TetR, the TetR promoter repressed less , and it results in an active gene expression of RFP (Figure 1C).

This BioBrick (BBa_1767003) was used as a device that allows to:

(1) To retrieve information about the expression of a fluorescent reporter downstream of the TetR promoter in absence and increasing concentrations of AHL.

(2) To retrieve information about the time that takes our system to start producing the florescent protein. By obtaining this information, we can decide how much time our bacteria will stay alive.

(3) To compare this BioBrick to a similar BioBrick that lack AiiA (BBa_K1767005).

The BioBrick that lacks AiiA should produce less RFP, because in presence of AHL , the Lux_AHL complex produces more TetR ( AHL degrades slower without AiiA ) , so the Lux promoter is activated more , TetR is more expressed which leads to a reduced activation of the TetR promoter and produces less RFP.

Experimental Setup

In order to test and compare these BioBricks an overnight starter culture of E.coli harboring the plasmids BBa_K1767003 or BBa_K1767005, respectively were diluted in a low-growth, low-autofluorescence BA buffer. AHL was added ranging from concentrations of 0.1 to 100000 nM and fluorescence measurements were taken every 30 minutes starting 120 min post-induction over range of 8-12 hours.

Results

As shown in Figure 3, fluorescence values of each time point were normalized by dividing fluorescence intensities by OD, and plotted as a function of time for three different inducer concentrations ( 100000nM, 100nM and 0nM).

Figure 3A

Figure 3B

As can be seen in Figure 3, both constructs (with (Figure 3B) and without (Figure 3A) AiiA) behave similarly in the first 200 minutes displaying increasing RFP expression. After 200 minutes, we can see that the construct without AiiA (Figure 3A) continues to show increasing expression of RFP for all three exemplarily shown AHL concentrations. Starting from approximately 450 min, a decrease followed by an increase of RFP expression can be observed. No difference in behavior can be observed for high (10000 nM), medium (100 nM) and no AHL concentrations. The construct harboring AiiA (Figure 2B) reaches a plateau of RFP expression after approximately 300 minutes which can be observed for all AHL values. A similar valley-like behavior as observed for the construct without AiiA of the fluorescence intensities can be seen.

Discussions

(1) For the first 300 minutes both bacterial strains (with and without AiiA) behave similarly as it is the time that takes the system to activate gene expression of inducible promoters, produce the proteins and maturation of the fluorescent protein. Hence, as the system is producing RFP in absence of AHL an increase in RFP is observed for the first 300 minutes.

(2) What has to be mentioned is that there is not significant difference in behavior for the bacterial strains without AiiA in presence (100-100000nM) and absence (0nM) of AHL. We were able to show (Figure 3A) that the pLux promoter can be activated with AHL concentration starting from approximately 1 nM. According to this data, we should be able to repress RFP expression of the genetic circuit, but this cannot be observed. Possible explanations might be the

a.leakiness of the pTet promoter that does not repress the system efficiently or

b.the slow degradation rate of RFP

In a recent publication of Qi and colleagues (Qi et al, 2013, Cell) demonstrated that activation and repression of a CRISPR/Cas-based system display a delay phase (approximately 10-50 minutes) and full repression or activation can be observed after 240 minutes.

Figure 4

As in our system we covered roughly 550 minutes of measurements, hence we should observe a difference of RFP expression levels in this period. In order to test both hypotheses we propose to do two things:

(1) test the pTet promoter as a single module in presence and absence of tetracycline.

(2) Exchange the “wildtype” RFP with a fluorescent gene fused to a ssR-degradation tag such as LVA or LAA. These tags have been frequently used in the iGEM community and genes encoding fluorescent proteins carrying a degradation can be found on the iGEM registry database.

The bacterial strain carrying the genetic circuit with AiiA shows again no difference in RFP expression levels in presence and absence of AHL. According to the results obtained from Figure 2B, in which no induction could be observed for all AHL concentrations at all time points, RFP should be constantly expressed and the system can be treated as “no AHL” present. However, as the two experiments for both genetic circuits with and without AiiA exhibit similar expression behaviors, more controls and experimental test need to be performed in order to present convincing conclusions.

Results 2:

Based on the results of the previous experiment (figure 3B and 3A ) and the mentioned graph from literature (Qi et al., 2013) we figured out that it might take longer for us to observe a change in fluorescence intensities hence we conducted another experiment with the same setup, but with a longer period (up to 720 min).

Figure 4B

Figure 4A

As can be seen we observe the same behavior of fluorescence values compared to figure 3A and 3B for the period until approximately 500 min . after this period both graphs for the bacterial strain with and without AiiA (Figure 4A and 4B, respectively) reach a plateau in which fluorescence values do not change significantly. For further information, see the discussions section of the previous experiment and the conclusion section below.

Conclusion and Outlook

In our future work, as mentioned in the course of the paragraphs, we plan to test and characterize

(1) Controllable activity of AiiA by

a. Varying RBS strength

b. Use of mutated versions of AiiA with reduced activity

c. Degradation tag on AiiA

(2) Test of TetR gene expression and pTet promoter

a. By testing TetR and its repression of the Tet promoter in presence and absence of tetracycline

(3) Modify genetic circuit

a. By exchanging RFP with several degradation tags

b. In case we observe a time-dependent RFP expression, exchange RFP with ccdB and further conduct experiments with ccdB

References

(1) Dong, Y.-H., Xu, J.-L., Li, X.-Z. & Zhang, L.-H. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci. 97, 3526–3531 (2000).

(2) Wang, L.-H. Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase). J. Biol. Chem. 279, 13645–13651 (2004).

(3) Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–83 (2013).

(4) Dong, Y.-H., Xu, J.-L., Li, X.-Z. & Zhang, L.-H. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci. 97, 3526–3531 (2000).

(5) Wang, L.-H. Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase). J. Biol. Chem. 279, 13645–13651 (2004).

(6) http://parts.igem.org/Lux

(7) http://parts.igem.org/Part:BBa_R0040

(8) http://www.uniprot.org/uniprot/P04483

(9) http://www.uniprot.org/uniprot/P62554

(10) https://www.wikigenes.org/e/gene/e/1238622.html