Plasmid

Part:BBa_K1453001

Designed by: Qiao Zhou, Renhe Luo   Group: iGEM14_SJTU-BioX-Shanghai   (2014-10-02)
Revision as of 12:07, 17 October 2014 by HoPe Hao (Talk | contribs) (→‎pBluescript II KS(+) EcoRV deletion)

pBluescript II KS(+) EcoRV deletion

Vector

We used pBluescript II KS(+) as our origin vector and then transformed it into the Connector of our desire. Pbluescript.jpg

Figure 1 Vector map of pBluescript II KS(+)

Connector

The so-called Connector is plasmid designed to bind with several different Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000). Connectees are fusion proteins who carry out functional enzymes and have TAL(Transactivator-like effectors) at one end that can bind to certain DNA sequences and you can design your own Connectees with different enzymes. For pBluescript II KS(+), we have selected three 14-nucleotide-long sequences called RSⅠ, RSⅡ and RSⅢ as the recognition sequences(RS). The recognition sequences must start with a T and end with a T. RS Ⅰ: TTCGATATCAAGCT RS Ⅱ: TGTGACTGGTGAGT RS Ⅲ: TTTGGTCATGAGAT ( RS Ⅰ contains partial restriction enzyme cutting site of EcoRI and EcoRV ) ( RS Ⅱ contains partial restriction enzyme cutting site of ScaI )


Figure 2 A Connector binds with three different Connectees

Figure 2 A Connector binds with three different Connectees

Combination

In order to prove that Connectors are able to bind with three kinds of Connectees. We designed the combination experiment. We selected a pathway: substrate becomes intermediate product through enzymeⅠ,then the intermediate product can either go to production A through enzymeⅡ or production B through enzyme Ⅲ.

SCAL11.png

Figure 3 Diagrams of our pathway design

To achieve this, we need three kinds of Connectees fused with enzyme Ⅰ, ⅡandⅢ independently and two kinds of Connectors. One has RSⅠand RSⅡ while the other has RSⅠand RSⅢ. So in the end, Connectors with RSⅠ and RSⅡ get production A while Connectors with RSⅠ and RSⅢ production B.


Scal14.png

Figure 4 Connector is originally designed with three recognition sequence(RS). Then we transformed it into two different Connector, one with RS Ⅰ and RS Ⅱ while the other one with RS Ⅰ and RS Ⅲ. Connector with RSⅠand RSⅡ can bind with Connectee-enzyme Ⅰ and Connectee-enzyme Ⅱ and get production A in the end, while Connector with RS Ⅰ and RS Ⅲ can bind with Connectee-enzyme Ⅰ and Connectee-enzyme Ⅲ and get production B. The corresponding Connectors are pBluescript II KS(+) ScaI deletion and pBluescript II KS(+) EcoRV deletion.

pBluescript II KS(+) EcoRV deletion

We delete the RSⅢ site so this transformed Connector can only bind to Connectees with two kinds of enzyme. In order to delete this site and surrounding sequence, we adopted the Inverse PCR to delete the following sequence: GGCTGCAGGAATTCGATATCAAGC

Primers for EcoRV:

Forward: TTATCGATACCGTCGACCTCG Tm:56℃

Reverse: CGGGGGATCCACTAGTTCTA Tm:53℃

pBluescript II KS(+) EcoRV deletion can be used along with pBluescriptIIKS(+) ScaI deletion and our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.

Inverse PCR

We used Inverse PCR to transform our plasmid into two kinds of Connectors: pBluescript II KS(+) ScaI deletion and pBluescript II KS(+) EcoRV deletion. Inverse PCR method is using cyclic DNA (such as a plasmid) as the template, with two primers designed in a reverse direction to achieve completed PCR. In this way, by designing primers,we can introduce a mutation, insertion or deletion. We used KOD-Plus-Mutagenesis Kit by TOYOBO.CO.LTD. More details please click http://www.bio-toyobo.cn.

Usage and Biology

If you want to use our Connector, there is a list for possible TAL recognition sequences on pBluescript II KS(+). You can choose any combination of following sequences to design your own Connectees.

'TAAAAAGGCCGCGT', 'TAAAAATGAAGTTT', 'TAAATCAAAAGAAT', 'TAAATCAGCTCATT', 'TAAATCGGAACCCT', 'TAAATTGTAAGCGT', 'TAACGCCAGGGTTT', 'TAACTCACATTAAT', 'TAAGATGCTTTTCT', 'TAAGCGTTAATATT', 'TAAGGGATTTTGGT', 'TAATACGACTCACT', 'TAATCAAGTTTTTT', 'TACCAATGCTTAAT', 'TACCCAGCTTTTGT', 'TACCGGATACCTGT', 'TACCTGTCCGCCTT', 'TACTCTTCCTTTTT', 'TACTGTCATGCCAT', 'TAGAACTAGTGGAT','TAGAAGGACAGTAT', 'TAGAGTAAGTAGTT', 'TAGATAACTACGAT', 'TAGCAGAGCGAGGT', 'TAGCGGTGGTTTTT', 'TAGCTGTTTCCTGT', 'TAGGGTTGAGTGTT', 'TAGGTATCTCAGTT', 'TAGTGAGGGTTAAT', 'TATATGAGTAAACT', 'TATCACTCATGGTT', 'TATCCGCTCACAAT', 'TATCCGGTAACTAT', 'TATCTCAGCGATCT', 'TATCTCAGTTCGGT', 'TATTATTGAAGCAT', 'TATTGGGCGCTCTT', 'TATTTGAATGTATT', 'TATTTTGTTAAAAT', 'TCAAAAAGGATCTT', 'TCAAAGGCGGTAAT', 'TCAACCAAGTCATT', 'TCAAGAAGATCCTT', 'TCAAGCTTATCGAT', 'TCAATCTAAAGTAT', 'TCACCAGCGTTTCT', 'TCACCTAGATCCTT', 'TCACGCTCGTCGTT', 'TCACGTTAAGGGAT', 'TCACTCATGGTTAT', 'TCACTGCCCGCTTT', TCAGCCCGACCGCT', 'TCAGCGATCTGTCT', 'TCAGCTCATTTTTT', 'TCAGTGAGGCACCT', 'TCATACTCTTCCTT', 'TCATAGCTCACGCT', 'TCATGGTCATAGCT', 'TCATTGGAAAACGT', 'TCCAGTCTATTAAT', 'TCCATCCAGTCTAT', 'TCCCATTCGCCATT', 'TCCCCCTGGAAGCT', 'TCCGCTTCCTCGCT', 'TCCGTAAGATGCTT', 'TCCTGCAACTTTAT', 'TCCTGTGTGAAATT', 'TCCTTTGATCTTTT', 'TCCTTTTTCAATAT', 'TCGATATCAAGCTT', 'TCGCCATTCAGGCT','TCGCGTTAAATTTT','TCGCTGCGCTCGGT','TCGGAAAAAGAGTT','TCGGCAAAATCCCT','TCGGGGCGAAAACT','TCGGTCGTTCGGCT', 'TCGGTGTAGGTCGT', 'TCGTCGTTTGGTAT', 'TCGTGCACCCAACT', 'TCGTGCGCTCTCCT', 'TCGTGTAGATAACT', 'TCGTTGTCAGAAGT', 'TCTAAAGTATATAT', 'TCTATCAGGGCGAT', 'TCTATTTCGTTCAT', 'TCTCAGCGATCTGT', 'TCTCAGTTCGGTGT', 'TCTCATGAGCGGAT', 'TCTCTTACTGTCAT', 'TCTGAGAATAGTGT', 'TCTGTCTATTTCGT', 'TCTTCAGCATCTTT', 'TGAACCATCACCCT', 'TGAAGTGGTGGCCT', 'TGAATACTCATACT', 'TGACAGTTACCAAT','TGACTCCCCGTCGT', 'TGACTCGCTGCGCT','TGAGAATAGTGTAT','TGAGCGCGCGTAAT','TGAGGCACCTATCT','TGAGTAAACTTGGT','TGAGTGAGCTAACT','TGATCCCCCATGTT', 'TGCAAAAAAGCGGT','TGCAAGCAGCAGAT','TGCACCCAACTGAT','TGCAGGAATTCGAT','TGCATAATTCTCTT','TGCCCGGCGTCAAT','TGCCGTAAAGCACT', 'TGCGCAACGTTGTT','TGCGCGCTTGGCGT','TGCGCTCGGTCGTT','TGCGCTCTCCTGTT',TGCGGCGACCGAGT','TGCTACAGAGTTCT','TGCTGAAGCCAGTT', 'TGCTGCAAGGCGAT','TGGAAAACGTTCTT','TGGCAGCACTGCAT','TGGCAGCAGCCACT', 'TGGCGCTTTCTCAT', 'TGGCGTTTTTCCAT', 'TGGTAGCGGTGGTT','TGGTAGCTCTTGAT', 'TGGTATGGCTTCAT', 'TGGTCATAGCTGTT', 'TGGTCCTGCAACTT', 'TGGTTTTTTTGTTT', 'TGTAACCCACTCGT', 'TGTCAGAAGTAAGT', 'TGTGAAATTGTTAT', 'TGTGACTGGTGAGT', 'TGTGTGAAATTGTT', 'TGTTGAATACTCAT', 'TGTTGAGATCCAGT', 'TGTTGTTCCAGTTT', 'TTAAAAATGAAGTT', 'TTAAAAGTGCTCAT', 'TTAAAATTCGCGTT', 'TTAAATCAGCTCAT', 'TTAATTGCGCGCTT', 'TTAGCTCCTTCGGT', 'TTATCAAAAAGGAT', 'TTATCACTCATGGT', 'TTATCAGGGTTATT', 'TTATCCGCCTCCAT', 'TTATTGAAGCATTT', 'TTCACCTAGATCCT', 'TTCCGCGCACATTT', 'TTCCTGTGTGAAAT', 'TTCGATATCAAGCT', 'TTCGCGTTAAATTT', 'TTCGGAAAAAGAGT', 'TTCGGTCCTCCGAT', 'TTGCGCAACGTTGT', 'TTGCTGGCGTTTTT', 'TTGGAAAACGTTCT', 'TTGGCCGCAGTGTT','TTGGGAAGGGCGAT','TTGGTATCTGCGCT', 'TTGGTCATGAGATT', 'TTGGTCTGACAGTT', 'TTGTAAGCGTTAAT', 'TTGTTAAATCAGCT', 'TTGTTCCCTTTAGT', 'TTGTTGCCATTGCT', 'TTTAAATTAAAAAT', 'TTTATCAGGGTTAT', 'TTTCACCAGCGTTT', 'TTTCCCCGAAAAGT', 'TTTCGTTCATCCAT', 'TTTCTACGGGGTCT', 'TTTCTGTGACTGGT', 'TTTGGAACAAGAGT', 'TTTGGGGTCGAGGT', 'TTTGGTCATGAGAT', 'TTTTAAATCAATCT', 'TTTTCAATATTATT', 'TTTTTCAATATTAT', 'TTTTTCCATAGGCT'

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 677
    Illegal SpeI site found at 683
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 683
    Illegal NotI site found at 669
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 689
    Illegal XhoI site found at 716
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 677
    Illegal SpeI site found at 683
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 677
    Illegal SpeI site found at 683
    Illegal NgoMIV site found at 328
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2089
    Illegal SapI site found at 1006


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Categories
Parameters
n/apBluescript II KS(+) EcoRV deletion