Difference between revisions of "Part:BBa K4156109"

 
 
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<partinfo>BBa_K4156109 short</partinfo>
 
<partinfo>BBa_K4156109 short</partinfo>
  
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pPepT-TP901-φ174E is a composite part that express lysis gene φ174E, constructed with hypoxia-sensing promoter pPepT and serine integrase TP901.
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<!-- Add more about the biology of this part here -->
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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We designed pPepT-TP901-φ174E to test the expression efficiency of φ174E under the control of a logic gate linking the pPepT (<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156078"> BBa_K4156078 </a></html>  ) and the serine integrase TP901 (<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156087"> BBa_K4156087 </a></html> ).We will validate the function of this biobrick by measuring the viability of the bacteria.
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===Characterization===
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==In vitro characterization and data analysis of the reported strains withφ174E==
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We constructed the lysis reporter CR by adding the pH-sensing promoter followed by the amplification genes Switch and mRFP. Fig 1 indicates the hypoxia (pPepT) inducing reporter after the addition of the lysis geneφ174E in induced and non-induced.The lower OD600 values indicate better lysis of the bacteria.The OD600 under anoxic conditions was lower than that under normoxic conditions, indicating that our constructed strain can respond well to the tumor environment.
 +
 
 +
Fig 2 indicates the fluorescence intensity of hypoxia (pPepT) induced reporter under induced and non-induced conditions after the addition of lysis gene φ174E.The fluorescence intensity under normoxic conditions was very low, while the fluorescence intensity under hypoxic conditions increased significantly after 8h.
 +
 
 +
Fig 3 is 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 anoxic 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.
 +
 
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/8-1.png" alt="control">
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                <figcaption><b>Figure 1:</b> The OD600 values over time by the CR reporter consisting of pPepT+φ174E+Switch+mRFP under hypoxic and normoxic conditions </figcaption>
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              </figure>
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</html>
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/8-2.png" alt="control">
 +
                <figcaption><b>Figure 2:</b> The fluorescence intensity over time by the CR reporter consisting of pPepT+φ174E+Switch+mRFP under hypoxic and normoxic conditions.</figcaption>
 +
              </figure>
 +
</html>
 +
 
 +
 
 +
<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/1917-3.png" alt="control">
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                <figcaption><b>Figure 3:</b>The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions under hypoxic and normoxic conditions.</figcaption>
 +
              </figure>
 +
</html>
 +
 
 +
 
 +
To further obtain the lysis-growth curve, we shortened the assay time to 5 min a measurement .  Fig 4, change in OD600 of hypoxia (pPepT)-induced reporter under induced and non-induced conditions. The results indicate that the lysis-growth curve is a dynamic function.
 +
 
 +
<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/8-6.png" alt="control">
 +
                <figcaption><b>Figure 4:</b>The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions at different pH values.</figcaption>
 +
              </figure>
 +
</html>
 +
 
 +
 
 +
Next, we tested the constructed CR reporters using CT26 cell cultures. In Fig 5 and 6, CT26 cells were cultured for 5 consecutive days, and the OD600 values and fluorescence response of the pPepT-controlled CR were tested. Fig 5 and 6, OD600 values and fluorescence response of pPepT-controlled CR tested using the above cell culture medium. Fig 5, OD600 values under anaerobic conditions were significantly smaller than those under normoxic conditions, and Fig 6 fluorescence values under anaerobic conditions were higher than those under normoxic conditions. The results indicate that CR reporters can respond in cell culture medium.
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 +
<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/8-7.png" alt="control">
 +
                <figcaption><b>Figure 5:</b>ig The OD600 values of pPepT-controlled CR based on CT26 cell medium samples under hypoxic and normoxic conditions.</figcaption>
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              </figure>
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</html>
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<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/8-8.png" alt="control">
 +
                <figcaption><b>Figure 6:</b>The fluorescence response of pPepT-controlled CR based on CT26 cell medium samples under hypoxic and normoxic conditions.</figcaption>
 +
              </figure>
 +
</html>
 +
 
 +
==Hypoxia-inducible promoter (pPepT) control effector engineered strains were co-incubated with RKO cells.==
 +
We linked pPepT-TP901 to XOR gate-HlyE （<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156119"> BBa_K4156119 </a></html>) for validation of treatment viability.
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Figure 7 shows the RKO cell activity after incubation with each strain ((OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, normoxic conditions. It can be seen that pPepT was not activated under normoxic conditions and the therapeutic protein was not expressed, so there was no significant change in RKO cell activity compared with the WT group.
 +
Figure8 shows the RKO cell activity of each strain after incubation in fresh DMEM medium, anoxic conditions. Under hypoxic conditions pPepT was activated and the expression of therapeutic proteins caused RKO cells to be killed, so RKO cell activity was significantly reduced compared to the WT group.
 +
 
 +
<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/11-1.png" alt="control">
 +
                <figcaption><b>Figure 7:</b>The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, anoxic conditions</figcaption>
 +
              </figure>
 +
 
 +
</html>
 +
<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/11-2.png" alt="control">
 +
                <figcaption><b>Figure 8:</b>The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in 3 day  DMEM medium,anoxic conditions</figcaption>
 +
              </figure>
 +
</html>
 +
 
 +
==Coincubation of different doses of effector engineered strains (OD=0.6) with RKO cells==
 +
We linked pPepT-TP901 to XOR gate-HlyE （<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156119"> BBa_K4156119 </a></html>) for validation of treatment viability.
 +
 
 +
Figure 9 shows the RKO cell activity after incubation with different doses of pPepT-controlled effector strains under fresh DMEM medium, anoxic conditions. It can be seen that RKO cell activity decreases with increasing doses of hypoxia-inducible promoter-controlled effector strains.
 +
<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/11-3.png" alt="control">
 +
                <figcaption><b>Figure 9:</b>The RKO cell activity after incubation with different doses of pPepT-controlled effector strains under fresh DMEM medium, anoxic conditions.</figcaption>
 +
              </figure>
 +
</html>
 +
==30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times==
 +
We linked pPepT-TP901 to XOR gate-HlyE （<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156119"> BBa_K4156119 </a></html>) for validation of treatment viability.
 +
 
 +
From the figure 10, it can be obtained that RKO cell activity decreased with increasing incubation time of the effector strains controlled by pPepT under fresh DMEM medium, anoxic conditions.
 +
<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/11-3.png" alt="control">
 +
                <figcaption><b>Figure 10:</b>The RKO cell activity decreased with increasing incubation time of the effector strains controlled by pPepT under fresh DMEM medium, anoxic conditions</figcaption>
 +
              </figure>
 +
</html>
 +
 
 +
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4156109 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4156109 SequenceAndFeatures</partinfo>

Latest revision as of 07:39, 13 October 2022


pPepT-TP901-φ174E

pPepT-TP901-φ174E is a composite part that express lysis gene φ174E, constructed with hypoxia-sensing promoter pPepT and serine integrase TP901.


Usage and Biology

We designed pPepT-TP901-φ174E to test the expression efficiency of φ174E under the control of a logic gate linking the pPepT ( BBa_K4156078 ) and the serine integrase TP901 ( BBa_K4156087 ).We will validate the function of this biobrick by measuring the viability of the bacteria.

Characterization

In vitro characterization and data analysis of the reported strains withφ174E

We constructed the lysis reporter CR by adding the pH-sensing promoter followed by the amplification genes Switch and mRFP. Fig 1 indicates the hypoxia (pPepT) inducing reporter after the addition of the lysis geneφ174E in induced and non-induced.The lower OD600 values indicate better lysis of the bacteria.The OD600 under anoxic conditions was lower than that under normoxic conditions, indicating that our constructed strain can respond well to the tumor environment.

Fig 2 indicates the fluorescence intensity of hypoxia (pPepT) induced reporter under induced and non-induced conditions after the addition of lysis gene φ174E.The fluorescence intensity under normoxic conditions was very low, while the fluorescence intensity under hypoxic conditions increased significantly after 8h.

Fig 3 is 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 anoxic 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 pPepT+φ174E+Switch+mRFP under hypoxic and normoxic conditions

control
Figure 2: The fluorescence intensity over time by the CR reporter consisting of pPepT+φ174E+Switch+mRFP under hypoxic and normoxic conditions.


control
Figure 3:The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions under hypoxic and normoxic conditions.


To further obtain the lysis-growth curve, we shortened the assay time to 5 min a measurement . Fig 4, change in OD600 of hypoxia (pPepT)-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 pH values.


Next, we tested the constructed CR reporters using CT26 cell cultures. In Fig 5 and 6, CT26 cells were cultured for 5 consecutive days, and the OD600 values and fluorescence response of the pPepT-controlled CR were tested. Fig 5 and 6, OD600 values and fluorescence response of pPepT-controlled CR tested using the above cell culture medium. Fig 5, OD600 values under anaerobic conditions were significantly smaller than those under normoxic conditions, and Fig 6 fluorescence values under anaerobic conditions were higher than those under normoxic conditions. The results indicate that CR reporters can respond in cell culture medium.

control
Figure 5:ig The OD600 values of pPepT-controlled CR based on CT26 cell medium samples under hypoxic and normoxic conditions.

control
Figure 6:The fluorescence response of pPepT-controlled CR based on CT26 cell medium samples under hypoxic and normoxic conditions.

Hypoxia-inducible promoter (pPepT) control effector engineered strains were co-incubated with RKO cells.

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

Figure 7 shows the RKO cell activity after incubation with each strain ((OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, normoxic conditions. It can be seen that pPepT was not activated under normoxic conditions and the therapeutic protein was not expressed, so there was no significant change in RKO cell activity compared with the WT group. Figure8 shows the RKO cell activity of each strain after incubation in fresh DMEM medium, anoxic conditions. Under hypoxic conditions pPepT was activated and the expression of therapeutic proteins caused RKO cells to be killed, so RKO cell activity was significantly reduced compared to the WT group.

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

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

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

Figure 9 shows the RKO cell activity after incubation with different doses of pPepT-controlled effector strains under fresh DMEM medium, anoxic conditions. It can be seen that RKO cell activity decreases with increasing doses of hypoxia-inducible promoter-controlled effector strains.

control
Figure 9:The RKO cell activity after incubation with different doses of pPepT-controlled effector strains under fresh DMEM medium, anoxic conditions.

30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times

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

From the figure 10, it can be obtained that RKO cell activity decreased with increasing incubation time of the effector strains controlled by pPepT under fresh DMEM medium, anoxic conditions.

control
Figure 10:The RKO cell activity decreased with increasing incubation time of the effector strains controlled by pPepT under fresh DMEM medium, anoxic conditions


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1624
  • 1000
    COMPATIBLE WITH RFC[1000]