Difference between revisions of "Part:pSB1C3"

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<partinfo>BBa_K1689004 short</partinfo>
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<partinfo>pSB1C3 short</partinfo>
  
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Nluc 398 fusion protein ORF
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pSB1C3 is a high copy number plasmid (RFC [10]) carrying chloramphenicol resistance.
  
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Firefly (Photinus pyralis) luciferase can be split to N terminal (Nluc) and C terminal (Cluc) fragments and each of them is inactive. When they two reassembled non-covalently, the enzymatic activity would be reconstituted and the recovered luciferase is able to oxidize luciferin and produce detectable bioluminescence. Currently there are different combinations of split fragments, among which Nluc 416 / Cluc 398 and Nluc 398/ Cluc 394 are widely used[1].
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The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).
  
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Rapamycin-binding domain (FRB) of human mTOR (mammalian Target of  Rapamycin) binds with high affinity to FK-506-binding protein 12 (FKBP). Rapamycin is able to induce the dimerization to form a FRB-rapamycin-FKBP complex[2]. This protein-protein interaction can be visualized by split luciferase[3]. FRB and FKBP are fused to Nluc and Cluc respectively, and adding rapamycin can induce the approaching and reconstitution of split luciferase (Figure 1a).
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pSB1C3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region.
  
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2015 Peking iGEM fused Nluc 398 to N terminus of FRB (Nluc 398-FRB, BBa_K1689004) and combined it with FKBP-Cluc 394 (BBa_K1689006) to validate the functional reconstitution of split luciferase. However, compared with Nluc 416/ Cluc 398, the bioluminescence intensity didn't increase significantly after rapamycin was added (Figure 1). Therefore we discarded them and chose Nluc 416/Cluc 398 as our split luciferase in the project (See BBa_K1689003 or BBa_K1689005).
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'''pSB1C3 is the designated Registry shipping plasmid backbone. All submissions for iGEM competitions must be submitted using pSB1C3.'''
  
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Go to [https://parts.igem.org/DNA_Submission_Instructions this page] for more information on how to ship submissions to iGEM HQ.
  
  
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===Usage and Biology===
  
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In Spring 2011, pSB1C3 was sequenced using several primers: Pre-R, Suff-F, VF2, VR, and internal primers for the origin and resistance. The reference sequence below matches the most recent sequencing verification of pSB1C3 (w/ BBa_J04450, located in SP 4000 Well 2A).
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[[File:Peking-FRB-FKBP-N3C3-2015-part-test.png|600px|]]
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'''Figure 1. Rapamycin-induced N-luc-FRB/FKBP-C-luc complementation. (a) The working mechanism of rapamycin induced dimerization. The interacting protein partners (FRB & FKBP) get closer and dimerize soon after rapamycin is added (40nM) [4], thus to reconstitute the enzymatic activity of luciferase. (b) The experimental data. Error bars denote s.d.; n=3.
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'''
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==References==
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1. Ramasamy Paulmurugan, Sanjiv S. Gambhir. Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals. Anal Chem. 2005 March 1; 77(5): 1295–1302.
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2. Rivera, V. M., T. Clackson, S. Natesan et al. A humanized system for pharmacologic control of gene expression.
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Nat. Med. 1996. 2:1028–1032.
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3. Ramasamy Paulmurugan, Sanjiv S. Gambhir. Combinatorial Library Screening for Developing an Improved Split-Firefly Luciferase Fragment-Assisted Complementation System for Studying Protein-Protein Interactions. Anal. Chem. 2007, 79, 2346-2353.
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4. Ramasamy Paulmurugan and Sanjiv S. Gambhir. Combinatorial Library Screening for Developing an Improved Split-Firefly Luciferase Fragment-Assisted Complementation System for Studying Protein-Protein Interactions. Anal. Chem. 2007, 79: 2346-2353.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1689004 SequenceAndFeatures</partinfo>
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<partinfo>pSB1C3 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1689004 parameters</partinfo>
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<partinfo>pSB1C3 parameters</partinfo>
 
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Revision as of 20:06, 18 September 2015

High copy BioBrick assembly plasmid

pSB1C3 is a high copy number plasmid (RFC [10]) carrying chloramphenicol resistance.

The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).

pSB1C3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region.

pSB1C3 is the designated Registry shipping plasmid backbone. All submissions for iGEM competitions must be submitted using pSB1C3.

Go to this page for more information on how to ship submissions to iGEM HQ.


Usage and Biology

In Spring 2011, pSB1C3 was sequenced using several primers: Pre-R, Suff-F, VF2, VR, and internal primers for the origin and resistance. The reference sequence below matches the most recent sequencing verification of pSB1C3 (w/ BBa_J04450, located in SP 4000 Well 2A).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2049
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 2055
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2049
    Illegal XhoI site found at 1033
    Illegal XhoI site found at 1925
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 2049
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 2049
    Plasmid lacks a suffix.
    Illegal XbaI site found at 2064
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.