四个改造质粒的part介绍：

1. pBlue script II KS(+) ScaI deletion

2 、 pBlue script II KS(+) EcoRV deletion

3 、 pBlue script II KS(+) 3 copy TTCGATATCAAGCT

4 、 pBlue script II KS(+) 5 copy TTCGATATCAAGCT

 

1. Vector

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

Figure 1 Vector map of pBluescript II KS(+)

 

This is the sequence of pBluescript II KS(+):

CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAG G^AA TT^C GAT^ATCAAGC T TATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGAT T TTGGTCATGAGA T TATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTC TGTGACTGGTGAGT^ACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

(Restriction site for EcoRI is marked with blue, restriction site for EcoRV is marked with red, restriction site for ScaI is marked with green. The TAL recognition sequences are highlighted with yellow while the start T base and end T base are grey.)

●List for possible TAL recognition sequences:

'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'

 

2.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

 

 

 

3.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.

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.

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(+) ScaI deletion：

We delete the RSⅡ 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:

CTGTGACTGGTGAGTACTCAACCAAGTCATTCTG

 

Primers for SacⅠ:

Forward: AGAATAGTGTATGCGGCGCGAC Tm:57℃

Reverse: AAAAGCATCTTACGGATGGCA Tm:58℃

 

pBluescript II KS(+) ScaI deletion can be used along with pBluescript II

KS(+) EcoRV deletion and our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.

 

●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 pBluescript II

KS(+) ScaI deletion and our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.

 

 

4.Maximization

We are trying to combine as many as enzymes as possible because more enzymes’ combination can produce more complicated reaction chains. So our purpose is to figure out the maximum number of Connectees that binding to a Connector.

So we intended to add more RS on the Connectors. On the one hand, more RSs mean we can bind more kinds of enzymes on one Connector. On the other hand ,we doubt the binding efficiency between Connectors and Connectees so more RSs can improve the possibility of Connectees binding to Connectors.

The corresponding Connectors are pBluescript II KS(+)_3_copy and pBluescript II KS(+)_5_copy.

 

●pBluescript II KS(+)_3_copy

In order to add 3 copies of RS on Connectors, we firstly use restriction enzyme BstXI and BamHI to make a nick and replace the original sequence with one RS. Secondly, in the same way, we use restriction enzyme SalI and KpnI to add another RS.

The RS sequence is TTCGATATCAAGCT.

Here we have designed the new short sequence:

and

This is the what we get in the end:

CT...CTCCACCGCGGTGGTTCGATATCAAGCTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACTTCGATATCAAGCTGGTACCCA….AC

pBluescript II KS(+)_3_copy can be used along with our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.

 

●pBluescript II KS(+)_5_copy

In order to add 4 more copies of RS on Connectors, we firstly use restriction enzyme BstXI and BamHI to make a nick and replace the original sequence with 4 consecutive repeats of RS.

The RS sequence is TTCGATATCAAGCT.

Here we have designed the new short sequence:

This is the what we get in the end:

CT…CTCCACCGCGGTGGTTCGATATCAAGCTTTCGATATCAAGCTTTCGATATCAAGCTTTCGATATCAAGCTGGATCCCC…AC

pBluescript II KS(+)_5_copy can be used along with our Connectees(iGEM12_SJTU_BioX_Shanghai BBa_K771000) to achieve your certain pathway design.

 

5.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.