Difference between revisions of "Part:BBa K3033014"
 
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<partinfo>BBa K3033014 AddReview 5</partinfo>
 
 
<I>University of Macau 2020</I>
 
<I>University of Macau 2020</I>
 
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<h3>UM_Macau 2020: Characterization of Magnetic ability improve</h3>
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<h3>UM_Macau 2020: Contribution: Enhancement of the magnetic properties of FtnA</h3>
  
 
<h4>Description</h4>
 
<h4>Description</h4>
  
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Inspired by the magnetization and control system from the UM_Macau team 2019, we design our bacteria cleaning system in the aquarium and make an improvement on their part. However, the magnetization capability of Ferritin is poorly limited. We have found literature that tagging iBox-PAK4 would be enhancing the magnetization capability of the FtnA protein.  
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Inspired by the magnetization and control system form UM_Macau team 2019, we designed our bacteria cleaning system for aquariums and make an improvement on their part. They overexpressed ferritin in E. coli to enhance bacterial iron storage capabilities, which allow them to control E. coli movement via magnetization.
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However, the magnetization capability of Ferritin is poorly limited. We have found literature which proves that tagging this iBox-PAK4 would be enhancing the magnetization capability of the FtnA protein. Ferritin is a ubiquitous iron storage protein. Thus, a larger protein assembly that contains more mineralized iron will generate larger response to magnetic forces.  
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InkaBox(BBa_K3630017)-PAK4(PAK4BBa_K3630018) can spontaneously form needle-like crystals when expressed in mammalian cells.
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Inka-PAK4, was originally described by Baskaran et. Al,  spontaneously forms needle-like crystals when expressed in mammalian cells.
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In this literature, researchers found that inkabox with PAK4cat undergoes conformational changes which allows the spontaneous crystallization of the complex, producing long rod-shaped crystals with a unit cell that has a hexagonal arrangement of subunits around a hollow channel (Fig. 1). Engineered ferritin-containing protein crystals, which named ftn-PAK4, exert magnetic forces that are 9 orders of magnitude larger than those in previous reports.
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literature show when inkabox with PAK4cat, conformational changes allow the spontaneous crystallization of the complex, producing long rod-shaped crystals with a unit cell that has a hexagonal arrangement of subunits around a hollow channel. The rod-shaped crystal exerts magnetic forces that are 9 orders of magnitude larger than those in previous reports.  
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       <img src="https://2020.igem.org/wiki/images/5/5a/T--UM_Macau--Crystalization_diagram.png" class= "center" width="800"
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       <img src="https://2020.igem.org/wiki/images/5/5a/T--UM_Macau--Crystalization_diagram.png" class= "center" width="400"
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<p style="text-align: justify; font-size: 14px; font-family: MuliLight; color: black; margin-left: auto; margin-right: auto;"><b>Figure 1.</b> Schematic of how ferritin subunits fit inside of the crystals’ hollow channel</p>
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<p style="text-align: justify; font-size: 14px; font-family: MuliLight; color: black; margin-left: auto; margin-right: auto;"><b>Figure 1.</b> Schematic of how ferritin subunits fit inside of the crystals’ hollow channel (Engineering a Genetically Encoded Magnetic Protein Crystal (2019))</p>
 
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For bacterial expression, Robert C. Robinson has several plasmids as expression vectors for Inka1 and PAK4, and successfully transfer to HEK293T cell.
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For bacterial expression, Robert C. Robinson has used pGEX4T1 (GE), pET28a (Novagen) and pSY5 (His tagged) as expression vectors for Inka1 and PAK4. Furthermore, the inka-PAK4 and GFP-inka-PAK4 plasmids have been transfected into HEK293T cells and have produced protein crystals successfully.
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Therefore, we linked this iBox-PAK4 encoding gene downstream of FtnA in our construct (Fig. 2). This hopefully enables our BREAC to better respond to magnetic forces applied to it and helps with their collection.
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Therefore, we encoding gene iBox(BBa_K3630017) and PAK4(PAK4BBa_K3630018) to downstream of the original FtnA(BBa_K3033014) and form the composite construct(BBa_K3630029) (fig. 2) to reach a stronger magnetic force.
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       <img src="https://2020.igem.org/wiki/images/2/23/T--UM_Macau--Ferritin_composite_sequence.png" class= "center" width="800"
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<p style="text-align: justify; font-size: 14px; font-family: MuliLight; color: black; margin-left: auto; margin-right: auto;"><b>Figure 2.</b> Optimized construct with iBox-PAK4</p>
 
<p style="text-align: justify; font-size: 14px; font-family: MuliLight; color: black; margin-left: auto; margin-right: auto;"><b>Figure 2.</b> Optimized construct with iBox-PAK4</p>
 
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Latest revision as of 15:00, 27 October 2020


FtnA

Derived from E.coli and synthesized through IDT. This codes for the ferratin which would be storing ferric ions for our magnetization function. A gene that originally contained in E.coli, which encodes for ferritin, a globular protein that consist of 24 subunits to form nanocage. The nanocage structure could store Fe3+ ions/Ferritin and therefore enhance the effectiveness and efficiency of E.coli magnetization for movement control.


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]


University of Macau 2020

UM_Macau 2020: Contribution: Enhancement of the magnetic properties of FtnA

Description

Inspired by the magnetization and control system form UM_Macau team 2019, we designed our bacteria cleaning system for aquariums and make an improvement on their part. They overexpressed ferritin in E. coli to enhance bacterial iron storage capabilities, which allow them to control E. coli movement via magnetization. However, the magnetization capability of Ferritin is poorly limited. We have found literature which proves that tagging this iBox-PAK4 would be enhancing the magnetization capability of the FtnA protein. Ferritin is a ubiquitous iron storage protein. Thus, a larger protein assembly that contains more mineralized iron will generate larger response to magnetic forces. Inka-PAK4, was originally described by Baskaran et. Al, spontaneously forms needle-like crystals when expressed in mammalian cells. In this literature, researchers found that inkabox with PAK4cat undergoes conformational changes which allows the spontaneous crystallization of the complex, producing long rod-shaped crystals with a unit cell that has a hexagonal arrangement of subunits around a hollow channel (Fig. 1). Engineered ferritin-containing protein crystals, which named ftn-PAK4, exert magnetic forces that are 9 orders of magnitude larger than those in previous reports.

HTML img Tag

Figure 1. Schematic of how ferritin subunits fit inside of the crystals’ hollow channel (Engineering a Genetically Encoded Magnetic Protein Crystal (2019))

For bacterial expression, Robert C. Robinson has used pGEX4T1 (GE), pET28a (Novagen) and pSY5 (His tagged) as expression vectors for Inka1 and PAK4. Furthermore, the inka-PAK4 and GFP-inka-PAK4 plasmids have been transfected into HEK293T cells and have produced protein crystals successfully. Therefore, we linked this iBox-PAK4 encoding gene downstream of FtnA in our construct (Fig. 2). This hopefully enables our BREAC to better respond to magnetic forces applied to it and helps with their collection.


HTML img Tag

Figure 2. Optimized construct with iBox-PAK4