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Backbone

Figure1. A backbone under constitutive promoter(pTetr). Has SlrP as a injection signal, GSK reporter, Bsa1 Cloning Site. Desired protein are inserted into the cloning site.

   Last year we used T3SS to inject GFP to mammalian cells. This year we wanted to explore T3SS limits. We submitted it to a injectable protein screening. We made a small library of proteins with distinguished structures which were chosen from 2011 Biobrick distribution.

   Assembling each protein, injection signal and reporter would have been a laborious task which we didn`t want to endure. So ready-to-inject backbones was proposed(Fig.1).

   We dsigned Bsa1 Cloning Site to facilitate quick assembly of proteins to inject. Thus it`s name ready-to-inject backbone. All inserts must be PCRed with specific primer to remove stop codon. Of course all inserts will be in-frame. See here for details.

   Another problem was how check if protein was injected. To solve it we used a distinct property of Glycogen Synthase Kinase 3 β, it is phosphorylated only in eucaryotic cells. This phosphorylation can by detected with phospho-specific antibodies. Thus we now can easily detect if protein was injected in eucaryotic cells. This property is exhibited by first 13aa of this protein^[1] which makes it very small reporter. See here for details.

   This design is one of the simpler ones. SlrP is an injection signal, without it the protein cannot be secreted. GSK is a reporter, by detecting phosphorylation of it you can distinguish whether it has been it eukaryotic cell. For us it is an evidence of successful injection. Bsa1 Cloning Site a neat cloning site which can be used for inserting various biobrick while retaining the whole constructs BioBrick properties. It is discussed bellow

Bsa1 Cloning Site

   Bsa1 Cloning site is unique in a sense that you can clone BioBrick into a middle of a construct and still retain the properties of biobrick. We used it to construct our backbones for T3SS characterization. Bsa1 cloning site is valuable part when you need to screen vast libraries of proteins. It designed that inserted biobrick would be fused to preceding signals.

   Bsa1 restriction enzyme is in distinguish group of enzyme which cutting site is different from recognition site. Unlike EcoR1 or Pst1, Bsa1 regognizes GGTCTC sequence but cuts the sequence 1 base further ahead of it. Which results in a 5 prime 4 base overhang(Fig).

5`...GGTCTCN^.......3`
3`...CCAGAGNNNNN^...5`

   You can manipulate the sequence of overhang as you like. By if you construct sequence GGTCTCNAATTN you can make it to ligate with Ecor1 digested strand. Also long as NAATTN won`t become GAATTG it wouldn`t not be digested by Ecor1 and that’s the beauty of it.

   Of course there are other restriction endonucleases that exhibit same properties but Bsa1 was the cheapest.

   However there are some limitations Bsa1 is not an official biobrick Restriction enzyme so you have to screen each part for Bsa1 recognition sequences. pSB1A3 has one in Ar locus which requires silent mutation or avoiding using it. Thus fur we didn`t encounter other BioBricks containing it.

   And because only backbone has to be digested by Bsa1 you don`t have to worry about inserts having Bsa1 sites.

   We designed a cloning site which when digested with Bsa1 will produce Not1 like overhang and Spe1 like overhang. We dealt with TAG stop codon at Xba1 site by insetting a mutation and destroying it. You can find how it is done in protocol section.

Reporter

   Glycogen Synthase Kinase 3 β is known to be phosphorylated by several enzymes in eukaryotic cell. We used first 13 amino acid sequence to construct a reporter which phosphorylation state could be detected. 9th amino acid which serine is phosphorylated in eukaryotic cell. There are antibodies which bind to only phosphorylated GSK reporter using them it is possible to distinguish whether it has been it eukaryotic cell. So you can see proteins which were injected into cell and which were not. This was a vital ingredient in our experiments.

References

1. Julie Torruellas Garcia, Franco Ferracci, Michael W. Jackson,1 Sabrina S. Joseph, Isabelle Pattis, Lisa R. W. Plano, Wolfgang Fischer, and Gregory V. Plano. 2006. Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag. Infect Immun.Vol.74:5645-57. [http://www.ncbi.nlm.nih.gov/pubmed/16988240 PubMed]

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