Difference between revisions of "Part:BBa K4960033"

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a novel payload loading Cre to verify recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells  
 
a novel payload loading Cre to verify recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells  
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===Profile===
 +
Name: Pdp1_NTD-3*GGSGG-Cre-GSSG-HiBiT,PAU_RS24015,PAU_RS16560,PAU_RS16565,PAU_RS16570<br>
 +
Origin: Synthetic<br>
 +
Properties: a novel payload loading Cre to verify recombinant PVCs can be reprogrammed to both<br>
 +
load and deliver non-native proteins into target cells <br>
  
 
<!-- Add more about the biology of this part here-->
 
<!-- Add more about the biology of this part here-->
 
===Usage and Biology===
 
===Usage and Biology===
 
For the delivery mediated by PVCs,Pdp1 NTD(the N-terminal domain (NTD) of Pdp1)(BBa K4960016) plays a significant role in loading diverse payloads(including the wild type and novel type) into the PVC complex as a ‘packaging domain’.Therefore,it is rebuilt by the NTD fusion with Cre(BBa K4960023) to  verify recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells .
 
For the delivery mediated by PVCs,Pdp1 NTD(the N-terminal domain (NTD) of Pdp1)(BBa K4960016) plays a significant role in loading diverse payloads(including the wild type and novel type) into the PVC complex as a ‘packaging domain’.Therefore,it is rebuilt by the NTD fusion with Cre(BBa K4960023) to  verify recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells .
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PAU_RS24015、PAU_RS16560、PAU_RS16565、 PAU_RS16570(BBa K4960017 BBa K4960018BBa K4960019BBa K4960020) may be involved in gene regulation and possibly contribute to the formation of PVCs in E.coil.<br>
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PAU_RS24015、PAU_RS16560、PAU_RS16565、 PAU_RS16570(BBa K4960017 BBa K4960018BBa K4960019BBa K4960020) may be involved in gene regulation and possibly contribute to the formation of PVCs in E.coil.[1]<br>
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Generally,this part is responsible for producing Cre Payload and loading it into PVC complex in subsequent experiment to confirm previous studies that recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells.<br>
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Generally,this part is responsible for loading Cre into PVC complex in subsequent experiment to confirm previous studies that recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells.<br>
  
 
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===Special Design===
 
===Special Design===
 
Fuse the NTD of Pdp1 with Cre(a novel protein for Payloads)<br>
 
Fuse the NTD of Pdp1 with Cre(a novel protein for Payloads)<br>
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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K4960033 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4960033 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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===Function===
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To demonstrate that Cre could work as our expectation, we transformed E01DARPin (pNC090, similar to the pAWP78-PVCpnf_pvc13-E01DARPin plasmid reported by Kreitz et al.) that specifically targets EGFR and a pPayload plasmid carrying Cre recombinase (pNC091, similar to the pBR322-Pdp1NTD-Cre-HiBiT plasmid reported by Kreitz et al.)into the engineered Escherichia coli.(Fig.1) Then PVCs were purified from E.coli electroporated with pNC090 (pPVC with EGFR targeting E01DARPin) and pNC091 (pPayload with Cre) via ultracentrifuge.(Fig.2) Negative-stain transmission electron microscopy resembled canonical PVC structures similar to the ones reported by Kreitz et al. (Fig.3), suggesting a successful assembly of PVCs.
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This part is used to load a novel protein for PVC named Cre into PVC.To demonstrate whether it can be properly loaded into PVC and sent into the cell to make a difference, we took the following methods:
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We incubated these EGFR-targeting, Cre-delivering PVCs with HEK-293T cells co-transfected with pLZ362 (PCMV-LoxP-STOP-LoxP-sNluc, a gift from Lihang Zhang, Westlake University) and either pNC089 (PCMV-EGFR) or pcDNA3.1(+) plasmids.Secreted Nanoluc (sNluc) levels in the culture medium were then evaluated at 72 h post PVCs treatment. Results showed a significant increase in sNluc levels in EGFR-expressing cells treated with PVCs, while the cells without EGFR expression showed low sNluc levels.These results demonstrated that Cre is correct and engineered PVCs could specifically deliver payload proteins into target mammalian cells, thereby serving as a possible delivery mechanism for our purposes.(Fig.4)
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We cotransformed the pPayload plasmid carrying this part and another plasmid E01DARPin (pNC090, similar to the pAWP78-PVCpnf_pvc13-E01DARPin plasmid reported by Kreitz et al.) that specifically targets EGFR into the engineered Escherichia coli.
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Figure 1
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Then PVCs were purified from E.coli electroporated with pNC090 (pPVC with EGFR targeting E01DARPin) and pNC091 (pPayload with Cre) via ultracentrifuge.('''Fig.1a''')<br>
 +
Negative-stain transmission electron microscopy resembled canonical PVC structures similar to the ones reported by Kreitz et al. '''Fig.1b'''), suggesting a successful assembly of PVCs.<br>
 +
We incubated these EGFR-targeting, Cre-delivering PVCs with HEK-293T cells co-transfected with pLZ362 (PCMV-LoxP-STOP-LoxP-sNluc, a gift from Lihang Zhang, Westlake University) and either pNC089 (PCMV-EGFR) or pcDNA3.1(+) plasmids.Secreted Nanoluc (sNluc) levels in the culture medium were then evaluated at 72 h post PVCs treatment. Results showed a significant increase in sNluc levels in EGFR-expressing cells treated with PVCs, while the cells without EGFR expression showed low sNluc levels.These results demonstrated that Cre is correct and engineered PVCs could specifically deliver payload proteins into target mammalian cells, thereby serving as a possible delivery mechanism for our purposes.('''Fig.1c''')<br>
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'''Figure 1.Validation of PVC Particles Specificity Delivery of Payload into Mammalian Cells. '''
 
<html>
 
<html>
  
 
<figure class="figure">
 
<figure class="figure">
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<img src="https://static.igem.wiki/teams/4960/wiki/composite-part/cre/cre-and-darpin-transformation.png"  height="200px">
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<img src="https://static.igem.wiki/teams/4960/wiki/composite-part/cre/2.jpg"  height="400px">
  
 
</figure>
 
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Figure 2
 
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<img src="https://static.igem.wiki/teams/4960/wiki/composite-part/cre/puri.png"  height="200px">
 
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Figure 3
 
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<figure class="figure">
 
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<img src="https://static.igem.wiki/teams/4960/wiki/composite-part/cre/.png"  height="200px">
 
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Figure 4
 
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<figure class="figure">
 
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<img src="https://static.igem.wiki/teams/4960/wiki/composite-part/cre/results.png"  height="200px">
 
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Figure 1.the Transformation of Cre and Darpin<br>
 
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Figure 2.Purification<br>
 
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Figure 3.PVC Structures under Electron Microscope<br>
 
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Figure 4.Result<br>
 
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===References===
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[1] Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F. Programmable protein delivery with a bacterial contractile injection system. Nature. 2023 Apr;616(7956):357-364.
 
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<partinfo>BBa_K4960033 parameters</partinfo>
 
<partinfo>BBa_K4960033 parameters</partinfo>
 
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Latest revision as of 05:27, 12 October 2023


pPayload promoter->Pdp1_NDT-3*GGSGG-Cre-GSSG-HiBiT->PAU_RS16570->PAU_RS16565->PAU_RS16560->PAU_RS240

a novel payload loading Cre to verify recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells

Profile

Name: Pdp1_NTD-3*GGSGG-Cre-GSSG-HiBiT,PAU_RS24015,PAU_RS16560,PAU_RS16565,PAU_RS16570
Origin: Synthetic
Properties: a novel payload loading Cre to verify recombinant PVCs can be reprogrammed to both
load and deliver non-native proteins into target cells

Usage and Biology

For the delivery mediated by PVCs,Pdp1 NTD(the N-terminal domain (NTD) of Pdp1)(BBa K4960016) plays a significant role in loading diverse payloads(including the wild type and novel type) into the PVC complex as a ‘packaging domain’.Therefore,it is rebuilt by the NTD fusion with Cre(BBa K4960023) to verify recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells . PAU_RS24015、PAU_RS16560、PAU_RS16565、 PAU_RS16570(BBa K4960017 BBa K4960018BBa K4960019BBa K4960020) may be involved in gene regulation and possibly contribute to the formation of PVCs in E.coil.[1]
Generally,this part is responsible for loading Cre into PVC complex in subsequent experiment to confirm previous studies that recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells.


Special Design

Fuse the NTD of Pdp1 with Cre(a novel protein for Payloads)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 910
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 596
  • 1000
    COMPATIBLE WITH RFC[1000]

Function

This part is used to load a novel protein for PVC named Cre into PVC.To demonstrate whether it can be properly loaded into PVC and sent into the cell to make a difference, we took the following methods: We cotransformed the pPayload plasmid carrying this part and another plasmid E01DARPin (pNC090, similar to the pAWP78-PVCpnf_pvc13-E01DARPin plasmid reported by Kreitz et al.) that specifically targets EGFR into the engineered Escherichia coli. Then PVCs were purified from E.coli electroporated with pNC090 (pPVC with EGFR targeting E01DARPin) and pNC091 (pPayload with Cre) via ultracentrifuge.(Fig.1a)
Negative-stain transmission electron microscopy resembled canonical PVC structures similar to the ones reported by Kreitz et al. Fig.1b), suggesting a successful assembly of PVCs.
We incubated these EGFR-targeting, Cre-delivering PVCs with HEK-293T cells co-transfected with pLZ362 (PCMV-LoxP-STOP-LoxP-sNluc, a gift from Lihang Zhang, Westlake University) and either pNC089 (PCMV-EGFR) or pcDNA3.1(+) plasmids.Secreted Nanoluc (sNluc) levels in the culture medium were then evaluated at 72 h post PVCs treatment. Results showed a significant increase in sNluc levels in EGFR-expressing cells treated with PVCs, while the cells without EGFR expression showed low sNluc levels.These results demonstrated that Cre is correct and engineered PVCs could specifically deliver payload proteins into target mammalian cells, thereby serving as a possible delivery mechanism for our purposes.(Fig.1c)
Figure 1.Validation of PVC Particles Specificity Delivery of Payload into Mammalian Cells.

References

[1] Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F. Programmable protein delivery with a bacterial contractile injection system. Nature. 2023 Apr;616(7956):357-364.