Part:BBa_K2500011

AND Gate B: Synthetic Promoter Responsive to LldR and LuxR

The AND gate integrates two signals, namely the presence of high L-lactate concentrations and high bacterial cell density (qorum sensing) and regulates the effector functions of CATE. It allows our engineered bacteria to autonomously decide whether they are currently located in tumor tissue or not.

In the absence of high concentrations of L-lactate, LldR inhibitor proteins bind to the operator sites O1 and O2 surrounding the pLux promoter leading to the formation of a DNA loop. The pLux promoter is sequestered and inaccessible for transcriptional activation by the quorum sensing components.

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]

Usage and Biology

Figure 1:Genetic circuit of CATE. CATE consists of an regulator (upper) and actuator (lower) plasmid. The AND gate integrates the inputs relayed by components encoded on the actuator plasmid and allows our bacteria to autonomously decide whether they are currently located in a tumor environment or not.

Input 1: L-Lactate

Excessive production of lactate even under normoxic conditions¹ is a common feature of cancer cells due to the well-known Warburg effect, which is why we selected this marker as one of the two inputs integrated by our AND gate. LldP is a L-lactate permease transporting L-lactate across the bacterial membrane. At low lactate concentrations, lldR repressor proteins bind the two operator sequences O1 and O2 surrounding the pLux quorum sensing promoter and oligomerize. The oligomerization of bound lldR proteins leads to the formation of a DNA-loop which sequesters the pLux promoter, making it inaccessible for transcription as depicted in Figure 2.

Figure 2: In the presence of lower concentrations of L-lactate, lldR repressor proteins bind the operator sequences of the AND gate surrounding the pLux promoter and prevent transcription of genes positioned downstream of this promoter.

Input 2: AHL (quorum sensing)

Quorum sensing allows bacteria to restrict the expression of certain genes to high bacterial cell densities. N-Acyl homoserine lactone (AHL) is a bacterial messenger molecule which is constantly produced at very low levels by LuxI, diffuses across cell membranes and binds transcriptional activator LuxR. The higher the cell density, the higher the concentration of AHL present in the surrounding of each cell. The AHL:LuxR complex together activate transcription of genes positioned downstream of pLux.

CATE is encoded in E. coli Nissle which has the intrinsic ability to survive and home in tumors.^{2 3} Therefore, an accumulation of bacteria is expected only within tumor cells which is why we chose cell density as the second input. In addition, the integration of quorum sensing also ensures that a sufficient and therefore deadly amount of bacteria carrying the cytotoxic agent have accumulated within the tumor, lowering the incidences of drug resistance due to the incomplete elimination of the tumor.

In conclusion, a combination of high concentrations of lactate and AHL is a unique molecular pattern that should only occur in tumor tissue and therefore represents the only site which can unlock the full cytotoxic capabilities of CATE.

Genetic design

We reasoned that flanking the pLux promoter with O1 and O2 should result in an AND-gate behavior and relied on the previously characterized parts BBa_K1847007 (O1-pconst-O2), BBa_R0062 (plux) and BBa_C0062 (LuxR).

In design B of the AND gate, we adapted the architecture of BBa_K1847007. The Anderson promoter was exchanged with the pLux promoter such that a) the -35 and -10 regions of the new promoter were at the same position as that of the original one and that b) the total distance between O1 and O2 was kept the same as in the original part.

Further, we duplicated the lldR binding sites O1 and O2 in order to achieve a molecular zipper mechanism which would lead to a tighter DNA-loop as more LldR repressors are bound. This design in turn requires even higher, tumor-specific concentrations of L-lactate to completely unzip the loop.

Figure 3: (Left) AND gate design B. LldR repressor binding sites O1 and O2 were duplicated to generate a molecular zip, which results in a tighter DNA-loop and thus should reduce leakiness of the promoter. (Right) The molecular zip mechanism recruits more LldR repressor proteins, shifting the AND gate activation point to higher lactate levels.

Characterization

We determined the dose-response behavior of our AND gates to the two inducers AHL and L-lactate and in order to assess whether our designs would be capable to distinguish healthy and tumor tissue with respect to lactate and expected AHL concentrations.

Two plasmids, a regulator plasmid and an actuator plasmid encoding AND-gate and sGFP, were transformed into E. coli TOP10 as shown in Figure 4. Exponential-phase cultures were induced in microtiter plates with combinations of 8 different AHL and 8 different L-lactate concentrations and analyzed after 5.5 hours growth in the plate. A detailed protocol is available at Protocols

Figure 4: (Top) Circuit used to characterize AND gate behaviors. (Bottom) Dose-response of different AND-gate designs to L-lactate and AHL. Shown are geometric means of two replicates of fold changes over non-induced case. b) Similar experiment, analysis and plot but with an increased range of concentrations of both inducers.

All our synthetic AND gates increase the production of sGFP with increasing inducer concentrations, whereby AND gate B clearly exhibits the lowest level of leakiness as expected due its molecular zipper. Hence, the highest level of activation coincides with the highest amounts of both inducers. No activation is observed at low and intermediary concentrations of inducers.

Modeling

AND gate activation model

Given our experimental results, we created a new model which considers the leakiness of both inputs, L-lactate and AHL, separately instead of combining them into a global leakiness to fit our model better to reality.

where

Fit of the AND gate promoter response

From the experimental data of the AND gate response, we could fit 3 parameters of our model: both leakinesses of our hybrid promoters with regard to LuxR-AHL and lactate, and half-activation concentrations of LuxR-AHL.

Figure 5: Fit of the activation of AND gate B. Parameter space fitting the experimental data. Each point represents a parameter vector that significantly fit the experimental data. The blue points fit the data the best (least sum of square) while the yellow ones represent parameters combinations that barely fit the data (but still significant according to the chi2 test of goodness of fit). Fitted parameters are annotated in red.

From our statistically significant fit and under the assumptions made, we were able to determine the following parameters for the AND gate:

Summary

• Our AND gate promoters allow CATE to distinguish levels of lactate and AHL in healthy tissue to those in tumor tissue
• AND gate B exhibits the lowest leakiness due to its molecular zipper mechanism
• We created a new model which realistically describes the behavior of the AND gate

References

1. F. Hirschhaeuser et al. "Lactate: a metabolic key player in cancer." Cancer Research (2011): 6921-6925
2. Y. Zhang et al. "Escherichia coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumors through Expression of Azurin Protein" Applied and Environmental Microbiology (2012): 7603–7610
3. J. Stritzker et al. "Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice." International Journal of Medical Microbiology (2007): 151-162