Difference between revisions of "Part:BBa K4156104"
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==Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells==
 
==Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells==
  
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Fig1 shows the activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in fresh DMEM medium,normoxic conditions, and it can be seen that the relative viability of RKO cells in the experimental groups with the addition of effector strains in fresh culture medium, except for the plac+HlyE positive control, did not change significantly compared with the WT group.
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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.
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Fig2 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.  
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                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-1.png" alt="control">
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                 <figcaption><b>Figure 1:</b>The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, normoxic conditions.</figcaption>
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                 <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, normoxic conditions.</figcaption>
 
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                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-2.png" alt="control">
 
                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-2.png" alt="control">
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                 <figcaption><b>Figure 2:</b> The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in 3 day  DMEM medium, normoxic conditions.</figcaption>
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                 <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, normoxic conditions.</figcaption>
 
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==Coincubation of different doses of effector engineered strains (OD=0.6) with RKO cells==
 
==Coincubation of different doses of effector engineered strains (OD=0.6) with RKO cells==
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Fig3 shows the RKO cell activity after incubation with different doses of plldR and pCadC control effector strains under3 day DMEM medium, normoxic conditions. The RKO cell activity decreased with increasing doses of effector strains. Fig4 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 decreased with increasing doses of effector strains controlled by hypoxic induced promoters.
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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.
 
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                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-3.png" alt="control">
 
                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-3.png" alt="control">
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                 <figcaption><b>Figure 3:</b>The RKO cell activity after incubation with different doses of plldR and pCadC control effector strains under 3 day DMEM medium, normoxic conditions..</figcaption>
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                 <figcaption><b>Figure 9:</b>The RKO cell activity after incubation with different doses of plldR and pCadC control effector strains under 3 day DMEM medium, normoxic conditions.</figcaption>
 
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==30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times==
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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.
 
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                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-4.png" alt="control">
 
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                 <figcaption><b>Figure 4:</b>The RKO cell activity after incubation with different doses of pPepT-controlled effector strains under fresh DMEM medium, anoxic conditions.</figcaption>
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                 <figcaption><b>Figure 10:</b>The RKO cell activity after incubation of plldR and pCadC control effector strains for different times under 3 day DMEM medium, normoxic conditions..</figcaption>
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==30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times==
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Fig5 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. From the fig6, 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.
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-5.png" alt="control">
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                <figcaption><b>Figure 5:</b> The RKO cell activity after incubation of plldR and pCadC control effector strains for different times under 3 day DMEM medium, normoxic conditions.</figcaption>
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                <figcaption><b>Figure 6:</b>  The RKO cell activity decreased with increasing incubation time of the effector strains controlled by pPepT under fresh DMEM medium, anoxic conditions</figcaption>
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Revision as of 02:50, 11 October 2022


pLldR-TP901

pLldR-TP901 is constructed with L-lactate-sensitive promoter pLldR and serine integrase TP901.


Usage and Biology

pLldR-TP901 consists of a fusion of the lactate-sensitive promoter pLldR and the serine integrase TP901. It will act as a complex regulatory for controlling downstream logic gates and transcription of genes.

Characterization

Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells

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.

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

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.

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

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..


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 614
    Illegal AgeI site found at 2743
  • 1000
    COMPATIBLE WITH RFC[1000]