Difference between revisions of "Part:BBa K4156101"

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Details of this section can be found in the next column "Addition of lysis genes"
 
Details of this section can be found in the next column "Addition of lysis genes"
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3.Lactate induced promoter-controlled effector engineered strain co-incubated with CT26 cells
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For the pLldR-Switch(TP901)-HlyE transformed effector, it was co-incubated with CT26 cells and ECN 1917 was used as a control. CT26 cells after incubation with different bacteria were assayed using Calcein/PI Cell Activity and Cytotoxicity Assay Kit (C2015S, Biotime) with wild-type E. coli 1917 as control and observed using fluorescence microscopy.
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The principle of the kit is that two probes can detect intracellular esterase activity and cell membrane integrity, respectively, thus reflecting cell activity and cytotoxicity. Calcein AM stains live cells with green fluorescence, while Propidium Iodide (PI) stains dead cells with red fluorescence.
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The results showed that the green fluorescence of cells stained by Calcein AM decreased significantly after co-incubation with the engineered bacteria, while the red fluorescence of cells stained by PI increased significantly(Fig 4). It was demonstrated that the cell activity was significantly reduced and the toxicity was significantly increased under the co-incubation of the engineered bacteria, i.e. the pLldR-Switch(TP901)-HlyE system had a significant effect on the treatment of tumor cells with CT26.
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/13-1.png" alt="control">
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                <figcaption><b>Figure4:</b>Fluorescence intensity of CT26 cells, co-incubated with effctors(pLldR-Switch(TP901)-HlyE) , versus control EcN 1917 .(CT26 were magnified 400-fold) </figcaption>
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==Addition of lysis genes==
 
==Addition of lysis genes==

Revision as of 07:21, 13 October 2022


pLldR

An Operon unit consisting of a collection of LldR-regulated promoters, and is capable of responding to lactate concentration.


Usage and Biology

pLldR is composed of the lldR regulatory protein ( BBa_K1847001 ), the terminator B0012, and the LldPRD promoter（ BBa_M36021 ）. At the lldprD promoter, there are two operator sites O1 and O2 , which are able to bind to the lldR protein and block subsequent gene transcription. The presence of lactic acid molecules can unblock this blocking phenomenon.[1-2] Thus, the pLldR can specifically respond to lactate and activate downstream genes. In our experiments, we used pLldR to adapt to the high lactate characteristics of the tumor cell microenvironment. Enabling our therapeutic strains to rapidly target to tumor cells and achieve targeted therapy.

Improvement

We built on the previously created parts lldPRD operon（ BBa_M36021 ) and lldR ( BBa_K1847001 ) by introducing a combination of hyper-spank and RBS spoVG in its head and an optimized sequence of RiboJ in its tail, once created a new pLldR promoter. Most importantly, we combined the constructed pLldR with the TP901-mediated XOR logic gate, which greatly improved the performance of pLldR.

The optimized pLldR promoter improves the signal output level while the signal output level is more stable. This is undoubtedly beneficial for the subsequent construction. More notably, we introduced the TP901-XOR gate system, which helps to coordinate many different inducible operons.We characterized our optimized system using pLldR-mRFP as a control as follows.

Characterization

Initial Testing of lactate Promoter

To Characterize part，we first added mRFP after the promoter and wanted to initially test the response of this promoter to different lactate concerntartions based on the fluorescence intensity. E. coli Nissle 1917 was used as chassis.Details of the characterization and test results can be found at BBa_K4156114

We constructed a lactate reporter consisting of the lactate-inducible promoter pLldR+mRFP. To test its performance, we added reporter in different chassis organisms.Fig 1 indicates that pLldR induces the expression of the downstream gene mRFP with the decrease of O2. Thus, it can be seen that the lactate reporter can work properly.

control
Figure1:Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+mRFP at different lactate concentrations.

Stability improvement

Then，amplifying genetic switches and Boolean logic gates based on serine integrase (TP901) are used in the design of biosensor systems [3]. These genetic devices enable bacteria to perform reliable detection, multiplex logic and data storage of clinical biomarkers in human clinical samples [4-5] to meet the requirements of medical testing. For characterization, we added switch, which is TP901 and XOR gate, then followed with mRFP. Details of the characterization and test results can be found at BBa_K4156104

1.In vitro characterization and data analysis of the reported strains

To improve signaling stability as well as accuracy, we added Amplifying genetic switches based on serine integrase (TP901) to the R reporter( BBa_K4156115 ) to construct the AR reporter. Fig 1 indicates lactate (plldR) induced AR reporter with homogenized fluorescence intensity (mRFP/Cell). Comparing Fig1, 2, it can be seen that the fluorescence intensity of the AR reporter decreased significantly at a lactate concentration of 0 mM, and its expression was more stable over time. The fluorescence intensity of the AR reporter was also greater at other concentrations of lactate induction, and the difference between the fluorescence intensity after lactate induction at each concentration was more pronounced. This result indicates that the addition of amplifying genetic switch enhances the reporter intensity and robustness of the lactate biosensor.

control
Figure 1: Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+Switch +mRFP at different lactate concentrations.

control
Figure2:Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+mRFP at different lactate concentrations.

We also observed the mRFP fluorescence intensity of WT 1917 and reporter strain AR(pLldR/pCadC/pPepT-Switch (TP901)-mRFP) after 48 h of induction using a fluorescence microscope (Olympus BX53). The results showed that the three promoters (pLldR, pCadC and pPepT)-Switch (TP901)-mRFP exhibited a uniform and clear red fluorescence signal after induction(Fig 3), indicating that the pLldR/pCadC/PepT-Switch (TP901) system could be expressed normally.

control
Figure3:Fluorescence intensity of engineered bacterias with pLldR/pCadC/pPepT-Switch (TP901)-mRFP , versus control EcN 1917 ,after 48h of induction.


2.Engineered strain co-incubated with RKO cells

Details of this section can be found in the next column "Addition of lysis genes"

3.Lactate induced promoter-controlled effector engineered strain co-incubated with CT26 cells

For the pLldR-Switch(TP901)-HlyE transformed effector, it was co-incubated with CT26 cells and ECN 1917 was used as a control. CT26 cells after incubation with different bacteria were assayed using Calcein/PI Cell Activity and Cytotoxicity Assay Kit (C2015S, Biotime) with wild-type E. coli 1917 as control and observed using fluorescence microscopy. The principle of the kit is that two probes can detect intracellular esterase activity and cell membrane integrity, respectively, thus reflecting cell activity and cytotoxicity. Calcein AM stains live cells with green fluorescence, while Propidium Iodide (PI) stains dead cells with red fluorescence.

The results showed that the green fluorescence of cells stained by Calcein AM decreased significantly after co-incubation with the engineered bacteria, while the red fluorescence of cells stained by PI increased significantly(Fig 4). It was demonstrated that the cell activity was significantly reduced and the toxicity was significantly increased under the co-incubation of the engineered bacteria, i.e. the pLldR-Switch(TP901)-HlyE system had a significant effect on the treatment of tumor cells with CT26.

control
Figure4:Fluorescence intensity of CT26 cells, co-incubated with effctors(pLldR-Switch(TP901)-HlyE) , versus control EcN 1917 .(CT26 were magnified 400-fold)

Addition of lysis genes

Because we have therapeutic proteins that cannot be exocytosed, it is not enough to simply stabilize the response signal, and we intend to add bacteriophage lysis gene phiX174E parts that will enable bacteria lysis.So next we added phiX174E to the above genetic parts. Details of the characterization and test results can be found at BBa_K4156105

1.In vitro characterization and data analysis of the reported strains withφ174E

We constructed the lysis reporter CR by adding the lactate-sensing promoter followed by the amplification genes Switch and mRFP( BBa_K4156114 ). Fig 1 indicates the lactate (plldR) inducing reporter after the addition of the lysis gene φ174E in induced and non-induced .The lower OD600 values indicate better lysis of the bacteria. It can be seen that the OD600 value becomes lower with increasing lactic acid concentration, and the OD value tends to a more stable state after 20 hour, indicating that our constructed strain can respond well to the tumor environment.

Fig 2 indicates the fluorescence intensity of lactate (plldR) induced reporters under induced and non-induced conditions after the addition of lysis gene φ174E. The fluorescence intensity showed an increasing trend with increasing lactate concentration.

Fig 3-5 are the OD600 of wild-type 1917 bacteria under induced and non-induced conditions, and the wild-type bacteria could hardly respond to the induction of lactic acid environments.

The results show that CR undergoes lysis under induced conditions, but the cells still produce fluorescence. It indicates that the fitted set of equations for lysis-growth should be a resonance function.

control
Figure 1:The OD600 values over time by the CR reporter consisting of pLldR+φ174E+Switch+mRFP at different lactate concentrations.

control
Figure 2: The fluorescence intensity over time by the CR reporter consisting of pLldR +φ174E Switch+ mRFP at different lactate concentrations.

control
Figure 3: The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions at different lactate concentrations.


To further obtain the lysis-growth curve, we shortened the assay time to 5 min a measurement . Fig 6, Changes in OD600 of lactate (plldR)-induced reporter under induced and non-induced conditions. The results indicate that the lysis-growth curve is a dynamic function.

control
Figure 4:The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions at different lactate concentrations.


Next, we tested the constructed CR reporters using CT26 cell cultures. In Fig 7 and 8, CT26 cells were cultured for 5 consecutive days, and the OD600 values and fluorescence response of the plldR-controlled CR were tested by measuring the lactate concentration after collecting the cell supernatant every 12 hours and using this sample as the medium; in Fig 7, as the lactate concentration in the culture increased, more bacteria were lysed and the OD600 values decreased accordingly. Fig 8, the fluorescence response profile was irregular. The results indicate that CR reporters can respond in cell culture medium.

control
Figure 5:ig The OD600 values of plldR-controlled CR based on the lactate concentration of CT26 cell medium samples.

control
Figure 6:The fluorescence response of plldR-controlled CR based on the lactate concentration of CT26 cell medium samples.

2.Lactate (pLldR) and pH (pCadC)Induced promoter-controlled effector engineered strain co-incubated with RKO cells

We linked pLldR-TP901--φ174E to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.

Figure 7 shows the RKO cell activity after incubation of each strain in fresh DMEM medium, normoxic conditions(OD=0.6, 30 μl, 3 hours). It can be seen that the RKO relative viability of the experimental groups with the addition of the effector strains in the fresh culture medium did not change significantly compared to the WT group, except for the plac+HlyE positive control.

Figure 8 shows the RKO cell activity of each strain after incubation in 3 day DMEM medium, normoxic conditions. It can be concluded that in the 3 day DMEM medium, due to the accumulation of metabolites such as cellular lactate, the lactate promoter and pH promoter were activated in the engineered strains and started to synthesize therapeutic proteins, resulting in a decrease in the relative viability of RKO compared to the WT group, especially in the pLldR+switch+HlyE and pCadC+switch+HlyE groups with the addition of the amplified gene switch. switch+HlyE group with the addition of the amplifying gene switch significantly reduced the RKO relative viability. In contrast, the decrease in RKO relative viability in the pLldR+φ174E+switch+HlyE group and pCadC+φ174E+switch+HlyE group was not significant, probably due to the decrease in the number of bacteria and the decrease in the number of synthesized therapeutic proteins by the addition of lysis genes.

control
Figure 7:The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, normoxic conditions.
control
Figure 8:The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in 3 day DMEM medium, normoxic conditions.

3.Coincubation of different doses of effector engineered strains (OD=0.6) with RKO cells

We linked pLldR-TP901--φ174E to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.

Figure 9 shows the RKO cell activity after incubation with different doses of plldR and pCadC control effector strains in 3 day DMEM medium, normoxic conditions. The RKO cell activity decreased with increasing doses of effector strains.

control
Figure 9:The RKO cell activity after incubation with different doses of plldR and pCadC control effector strains under 3 day DMEM medium, normoxic conditions.
4.30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times

We linked pLldR-TP901--φ174E to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.

Figure 10 shows the RKO cell activity after incubation of plldR and pCadC control effector strains for different times under 3 day DMEM medium, normoxic conditions. It can be seen that the RKO cell activity decreased with the increase of co-incubation time.

control
Figure 10:The RKO cell activity after incubation of plldR and pCadC control effector strains for different times under 3 day DMEM medium, normoxic conditions..

Applications

1.Western blot

To verify the extracellular secretion of HlyE, We connect pLldR to HlyE and constructed an AE strain by fusing his tag at the C-terminus of HlyE. Then, the AE strain (HlyE with his tag) was inoculated in 50 ml of LB medium containing the corresponding antibiotics and cultured overnight at 37 °C. Then, 5 ml of the culture was centrifuged and the supernatant was collected. The supernatant was concentrated using the TCA precipitation method (25% TCA, -20°C, 1h) to isolate the total protein. Finally, the expression of HlyE was detected by western blot. The results showed that the constitutive promoter could secrete HlyE under both inducible and non-inducible conditions, while the lactate (plldR), pH (pCadc) and hypoxia (pPepT) inducible reporters could only secrete HlyE under inducible conditions and not under non-inducible conditions. indicated that our constructed AE strain could well cope with environmental induction and secrete HlyE in the tumor microenvironment It was shown that our AE strain could respond well to environmental induction and secrete HlyE in the tumor microenvironment, thus killing cancer cells without harming other normal cells.

References

1 Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. Apr 2008;190(8):2997-3005. doi:10.1128/jb.02013-07

2 Weghoff MC, Bertsch J, Müller V. A novel mode of lactate metabolism in strictly anaerobic bacteria. Environ Microbiol. Mar 2015;17(3):670-7. doi:10.1111/1462-2920.12493

3 Courbet A, Endy D, Renard E, Molina F, Bonnet J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med. May 27 2015;7(289):289ra83. doi:10.1126/scitranslmed.aaa3601

4 Benenson Y. Biomolecular computing systems: principles, progress and potential. Nat Rev Genet. Jun 12 2012;13(7):455-68. doi:10.1038/nrg3197

5 Bonnet J, Yin P, Ortiz ME, Subsoontorn P, Endy D. Amplifying genetic logic gates. Science. May 3 2013;340(6132):599-603. doi:10.1126/science.1232758

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 688
  • 1000
    COMPATIBLE WITH RFC[1000]