Controlling the activation of the lytic cycle of lambda bacteriophage using a cI-RecA730 based genet

Usage and Biology

We designed and tested a genetic circuit for the controlled activation of the lytic cycle in lambda phages upon an external input.
In lambda phage the lambda repressor cI controls the transition between the lysogenic and lytic cycle. When present in high concentrations, cI represses the expression of cro and the phage stays in the lysogenic cycle (Figure 1). To de-repress cro expression and thus shift the phage in the lytic cycle, cI is proteolytically cleaved by RecA. We aim to use this system and control it by an external input, e.g., by adding the common inducing agent IPTG to the culture. Since DNA damage is required for RecA to be active we use a constitutively active mutant of RecA, RecA730. This protein has a point mutation (E38K)^[1], which causes RecA730 to be in its active form as soon as it is expressed. We hypothesized that directly controlling recA730 expression and at the same time decreasing cI expression, shall lead a strong de-repression of cro and thus the activation of the lytic cycle in lambda phage. To achieve this intention, we use a T7 phage promoter to control recA730 and tetR expression by external addition of IPTG (Figure 2), and eventually transformed the construct in the T7 system compatible expression strain E. coli BL21 (DE3). At the same time, we set the expression of cI under the control of a tetO operator which is repressing the expression of the downstream gene upon binding of its specific transcription factor TetR. Consequently, RecA730 will cleave remaining cI, TetR represses the expression of new cI, and cI cannot repress cro expression which leads to the activation of the lytic cycle in the lambda phage. We decided to evaluate the genetic circuit by using P_R promoter which natively regulates cro expression, for expressing the fluorescent reporter protein eGFP. Accordingly, when the circuit is functional and cI level within the phage is low, the level of eGFP will increase upon de-repression upon cI cleavage.

Figure 1. The shift between lysogenic and lytic state is toggled by the operator region of the lambda phages genetic switch. a. The operator consists of three parts (O_R1, O_R2, O_R3) overlapping with the promoters PRM and PR. PRM enables the expression of cI (lysogenic state) and P_R enables the expression of cro (lytic state). b. cI dimers are binding to O_R1 and O_R2, repressing the Cro production and enabling an optimal cI production by enhancing the binding affinity of the RNA-polymerase to P_RM. Thus, the lambda phage stays in the lysogenic cycle. c. Cro dimers bind to O_R3 and the production of cI is repressed, initiating the lytic cycle. Modified after Ptashne 2004.^[2]

Figure 2. Schematic overview illustrating the design of our genetic circuit for phage induction. a. General order of the genetic elements in our construct. b. Our genetic circuit before induction through IPTG: repression of the egfp expression through expression of cI, which is representative for the phage being kept in lysogenic state. c. Our genetic circuit after induction with IPTG: expression of recA730, cleaving cI and expression of tetR, which represses the expression of cI. This leads to eGFP production which is representative for the phage switching to lytic state.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Results

Assembly

The successfully cloned plasmids were further sequenced. The sequencing results showed two mutations in the 3741 bp sequence. One is placed in the terminator sequence after tetR. This should not impose any problems since it only brings in an additional CG-base pair in a non-coding region without a recognition site. The second mutation on the other hand is located in the coding region of the lambda repressor cI. The amino acid Gly-54 is replaced by Asp-54. Since the new amino acid features a negatively charged side chain, this could result in a different folding behavior of one of the key enzymes in our genetic switch. However, we assume that the mutation has little or no effect on the binding affinity of cI to the P_R promoter, since the following assay showed the expected results.

Fluorescence Assay

The phage induction module was characterized by measuring eGFP fluorescence of the respective culture before and after the induction with IPTG. For this we monitored the OD600 and the fluorescence of eGFP [extinction 485 nm / emission 528 nm]. We further tried to also decrease the fluorescence level by de-repressing TetR repression of cI expression by addition of anhydrotetracycline (AHT). AHT specifically binds to TetR, triggers its allosteric shift and thus its unbinding from tetO. Consequently, in the presence of AHT, the repression of cI expression is lifted and eGFP expression in turn is repressed by cI.
Ideally, the circuit will be engineered for induction by the quorum sensing molecule AHL from Pseudomonas aeruginosa in the future.
As expected, the ratio of fluorescence per optical density does increase rapidly after the induction (195 min) with IPTG.

Figure 3. Fluorescence intensity normalized to the optical density at 600 nm of our sleeper cell before and after the induction with IPTG. The measurements were carried out at 30 min intervals. Between these measuring points the samples were shaken at 225 rpm and 37°C. The induction with 0.5 mM IPTG took place after 195 min. Two biological replicates and five technical replicates per sample per measuring point have been measured.

As a result, a nearly 12-fold increase in this regard was observed, strongly indicating that our composite part does work as intended. Furthermore, sample 12 without induction showed a stable baseline over a timeframe of more than 20h. The behavior of sample 13 seems to be the result of a low optical density, since the raw fluorescent output is not much higher than the blank background. This suggest that our system is working with little to no significant basal expression. Nevertheless, it is necessary to mention that the informative value of our analysis is limited by the number of samples. While we did test biological duplicates, our number of technical replicates amounts to five per sample per measuring point. This has been conducted two times.
Also, we wanted to strengthen our assumption, that these observed effects are achieved through our construct. Therefore, we inhibited the system through addition of anhydrotetracycline (AHT).

Figure 4. Fluorescence intensity normalized to the optical density at 600 nm of our sleeper cell before and after the induction with IPTG. The measurements were carried out at 20 min intervals. Between these measuring points the samples were shaken at 225 rpm and 37°C. The induction with 0.5 mM IPTG took place after 146 min. The inhibitions took place after 146 min (t1) and 214 min (t2). Two biological replicates and five technical replicates per sample per measuring point. a. Inhibition assay of P12. b. Inhibition assay of P13.

There is a visible difference between the P12+IPTG sample and the P12+IPTG+AHT t1 sample, which was inhibited at the same point as the induction took place (146 min). This is true for both biological replicates. In both cases the discrepancies are greater than their respective error margins. On the other hand, when inhibited later in the measurement (214 min), the effect is less striking. While there is a difference in the P12 samples, it is not outside the error margin. With P13, the measurements are nearly identical. The fitted curve indicates, that the AHT addition after 214 min leads to an increased expression of egfp. However, this is probably the consequence of the two measurement points after 367 min and 387 min, which can be explained by measurement errors. The samples (P12-P13) inhibited at t1 achieved only 53 % of the uninhibited fluorescence output after about 50min upon induction. In contrast, the P12-P13 mean maximum fluorescence when inhibited at t1 is about 80 % of the regular induced sample. Overall, the comparison of the induced sample and the direct inhibition indicates that TetR was bound by AHT, which resulted in the renewed expression of cI. This shifts the cI-RecA730 equilibrium and results in a decreased expression of egfp. It is worth noticing, that even when inhibited at the same time as the induction occurs, the fluorescence output is still increasing at a greater speed than the optical density. This implies, that our switch and the respective assembly could be further simplified by erasing the TetR component, if one is willing to trade about 30 min of reaction time in the system. This could serve as a starting point for further engineering using an iterative approach to optimise our genetic switch.

Conclusion

In conclusion, we were able to gather data which matched our expectations in nearly every sample including the controls. The only measurement which was unexpected was the little difference between the second point of inhibition compared to the induced sample without AHT. This makes us optimistic about the further characterizations and applications of our composite part at a later point in time. A logical next step would be the induction of our circuit in a transformed lysogen. For further development RecA730 expression could be triggered by quorum sensing mechanims as shown in our Pathogen Sensing genetic circuit (see our wiki.)

References

[1]: Vlašić I, Šimatović A, Brčić-Kostić K. Genetic Requirements for High Constitutive SOS Expression in recA730 Mutants of Escherichia coli. Journal of Bacteriology. 2011 Sep 15:4643–4651. http://dx.doi.org/10.1128/JB.00368-11

[2]: Ptashne M. Chapter One: The master elements of control. In: A Genetic Switch: Phage Lambda Revisited. 3rd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2004. p. 160.