Difference between revisions of "Part:BBa K3505043"

 
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<partinfo>BBa_K3505043 short</partinfo>
 
<partinfo>BBa_K3505043 short</partinfo>
  
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<b>This backbone combined with the insert <bbpart>BBa_K3505041</bbpart> results in this comlpete Vector <bbpart>BBa_K3505007</bbpart></b> </b>
  
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Level 0 vector for Golden Braid assembly. The Level 0 modules are Interchangable parts in order everyone can use it as it is without any modification based on Golden Braid grammar.LacZa module as cargo for Blue White Screening between 2 BsmBI sites and 2 BtgzI sites. Resistance in Chloramphenicol and ori pMB1for high replication of DNA.
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    [[Image:T--Thessaly--GBcloninggrammar.jpeg|900px|thumb|none|]]
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<b>Figure 1.</b> The GB2.0 grammar
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    [[Image:T--Thessaly--pudp2.png|700px|thumb|none|]]
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<b>Figure 2.</b> pUPD2 map. Features and Restriction Sites.
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    [[Image:T--Thessaly--pudp2 map.png|700px|thumb|none|]]
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<b>Figure 3.</b> pUPD2
 
===Usage and Biology===
 
===Usage and Biology===
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GoldenBraid (GB) is a DNA assembly strategy for Plant Synthetic Biology based on Type IIS enzymes. It is also compatible for MoClo assembly.
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The sequences must not contain BsmBI and BsaI sites! Domestication may be done in order to vanish BsaI and BsmBI sites from the inner sequence.
  
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GB proposes an alternative view of modular cloning, and essentially the change is that you can infinitely build assemble new vectors by performing “braids”.[1] Using BsmBI  another big advantage is the use of a single level 0 vector (pUPD and pUPD2, where pUPD2 is derived from iGEM-borne pSB1C3) for any GBpart one needs.
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<span class='h3bb'>Sequence and Features</span>
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Then combining the desirable fragments from level 0; ‘level alpha’ (level a) cloning is succeeded creating Transcription Units (TU). Desirable TUs are combined to result in (Level Ω) cloning.
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* Using BsmBI for Level 0 modules and ‘level omega’ (Level Ω)
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* Using BsaI for ‘Level alpha’ (Level a)
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But this is not the end, GoldenBraid outperforms GoldenGate (which everyone knows) because of the ability to continuously clone TUs in an “exponential”  manner, compared to the linear progression GoldenGate. Bianry assembly of 2 Level Ω (Level 2 for MoClo) result in an alpha vector. Again Binary assembly of 2 alpha  result in an omega vector. With this assembly you can insert step by step as many parts and as many TUs you want with high efficiency.
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=== Experimental Use and Experience===
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The following parts were cloned in this vector
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<bbpart>BBa_K3505003</bbpart>
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<bbpart>BBa_K3505005</bbpart>
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<bbpart>BBa_K3505006</bbpart>
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<bbpart>BBa_K3505012</bbpart>
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<bbpart>BBa_K3505013</bbpart>
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<bbpart>BBa_K3505014</bbpart>
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<bbpart>BBa_K3505015</bbpart>
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<bbpart>BBa_K3505016</bbpart>
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<bbpart>BBa_K3505017</bbpart>
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<bbpart>BBa_K3505018</bbpart>
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<bbpart>BBa_K3505019</bbpart>
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<bbpart>BBa_K3505020</bbpart>
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<bbpart>BBa_K3505021</bbpart>
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<bbpart>BBa_K3505022</bbpart>
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<bbpart>BBa_K3505023</bbpart>
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<bbpart>BBa_K3505024</bbpart>
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==pUPD2 verification with no insert==
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[[File:T--Thessaly--pudp2ggel.png|500px|thumb|none|<i><b>Fig.4:</b> pUPD2 with no insert digested with EcoRI. Expected bands 2414bp, 276bp </i>]]
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===Sequence and Features===
 
<partinfo>BBa_K3505043 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3505043 SequenceAndFeatures</partinfo>
  
  
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===Functional Parameters===
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===References===
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<partinfo>BBa_K3505043 parameters</partinfo>
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[1] Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez (2013). “GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology.” <em>Plant Physiology</em> , 162 (3) 1618-1631; DOI: 10.1104/pp.113.217661

Revision as of 08:29, 24 October 2020


pUPD2 BACKBONE for Golden Braid Cloning

This backbone combined with the insert BBa_K3505041 results in this comlpete Vector BBa_K3505007 </b>

Level 0 vector for Golden Braid assembly. The Level 0 modules are Interchangable parts in order everyone can use it as it is without any modification based on Golden Braid grammar.LacZa module as cargo for Blue White Screening between 2 BsmBI sites and 2 BtgzI sites. Resistance in Chloramphenicol and ori pMB1for high replication of DNA.


T--Thessaly--GBcloninggrammar.jpeg

Figure 1. The GB2.0 grammar


T--Thessaly--pudp2.png

Figure 2. pUPD2 map. Features and Restriction Sites.

T--Thessaly--pudp2 map.png

Figure 3. pUPD2

Usage and Biology

GoldenBraid (GB) is a DNA assembly strategy for Plant Synthetic Biology based on Type IIS enzymes. It is also compatible for MoClo assembly. The sequences must not contain BsmBI and BsaI sites! Domestication may be done in order to vanish BsaI and BsmBI sites from the inner sequence.

GB proposes an alternative view of modular cloning, and essentially the change is that you can infinitely build assemble new vectors by performing “braids”.[1] Using BsmBI another big advantage is the use of a single level 0 vector (pUPD and pUPD2, where pUPD2 is derived from iGEM-borne pSB1C3) for any GBpart one needs.

Then combining the desirable fragments from level 0; ‘level alpha’ (level a) cloning is succeeded creating Transcription Units (TU). Desirable TUs are combined to result in (Level Ω) cloning.

  • Using BsmBI for Level 0 modules and ‘level omega’ (Level Ω)
  • Using BsaI for ‘Level alpha’ (Level a)

But this is not the end, GoldenBraid outperforms GoldenGate (which everyone knows) because of the ability to continuously clone TUs in an “exponential” manner, compared to the linear progression GoldenGate. Bianry assembly of 2 Level Ω (Level 2 for MoClo) result in an alpha vector. Again Binary assembly of 2 alpha result in an omega vector. With this assembly you can insert step by step as many parts and as many TUs you want with high efficiency.

Experimental Use and Experience

The following parts were cloned in this vector BBa_K3505003 BBa_K3505005 BBa_K3505006 BBa_K3505012 BBa_K3505013 BBa_K3505014 BBa_K3505015 BBa_K3505016 BBa_K3505017 BBa_K3505018 BBa_K3505019 BBa_K3505020 BBa_K3505021 BBa_K3505022 BBa_K3505023 BBa_K3505024

pUPD2 verification with no insert

Fig.4: pUPD2 with no insert digested with EcoRI. Expected bands 2414bp, 276bp

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 2061
    Illegal SpeI site found at 14
    Illegal PstI site found at 28
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2061
    Illegal SpeI site found at 14
    Illegal PstI site found at 28
    Illegal NotI site found at 21
    Illegal NotI site found at 2067
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2061
    Illegal XhoI site found at 1045
    Illegal XhoI site found at 1937
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 2061
    Illegal suffix found in sequence at 14
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 2061
    Illegal XbaI site found at 2076
    Illegal SpeI site found at 14
    Illegal PstI site found at 28
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez (2013). “GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology.” Plant Physiology , 162 (3) 1618-1631; DOI: 10.1104/pp.113.217661