Intro

We further developed "Dr. E. coli": our project of iGEM 2010. Last year, we showed that Type 3 Secretion System (T3SS) works in E. coli by injecting GFP into RK13 cells. We thought this system can be applied to direct reprogramming of somatic cells among many other things.

See [here] for details.

This year we repeated previous competitions experiment, one more time showing that GFP can be really injected into a target cell with it. And then, as a second step, we tested T3SS performance and tried to make it more convenient. For this purpose we designed a plasmid backbone which can instantly produce ready-to-inject fusion proteins from ordinary biobrick part. Using it, we tried to further characterize this system by injecting characteristic proteins. Type 3 Secretion System

T3SS is a system of pathogenic gram-negative bacterium such as Salmonella, Yersinia and EPEC (entero pathogenic E. coli). Using this system bacteria can inject whole protein molecules through a syringe like organelle named T3S Apparatus. During iGEM 2010 we found that E. coli with a part of Salmonella genome library expresses T3SS functionally. This presented opportunity to work with the amazing machinery without involving pathogenic bacteria. Read more

Plasmid Backbone for protein injection

We developed plasmid backbone which can attach tags needed for secretion and various other functions to a chosen protein biobrick. This can be used for big scale screening of various protein domains for their inject-ability. Backbone

Figure1. A backbone under constitutive promoter(pTetr). Has SlrP as a injection signal, GSK reporter, Bsa I 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. Because of it`s small size the interference on tag proteing should be minimu. See here for details.

We combining these futures and constructed ready-to-inject bakcbone. 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 is used for inserting various BioBrick while retaining the whole constructs BioBrick properties. The whole proteins is under control of pTetr constitutive Promoter.

With ready-to-inject backbone we were able to screen 8 distinct members of protein family. Carying different domains and performing various functions. We chose them from already available BioBrick distributions. Which means that they are of significant importance for iGEM. They are: mnt repressor, Gal4 - DNA binding domain (Zn2/Cys6), RFP, GFP, Cre DNA recombinase, (CCR5) transmembrane, LacI, Luciferase. See here for details. Resuls

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, Bsa I 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 EcoR I 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 (Fig). Which will ligate to Not1 and Spe1 but won't be digested after.

         Bsa1    Not1`           Spe1`   Bsa1
  
5`...GG GGTCTC A^GGCC ….........^CTAG A GAGACC...3`
3`...CC CCAGAG T CCGG^TCCGGCCGCT GATC^T CTCTGG...5`

5`...GG GGTCTC A                 CTAG A GAGACC...3`
3`...CC CCAGAG T CCGG                 T CTCTGG...5`

We dealt with TAG stop codon at Xba1 site by inserting a mutation and destroying it. Relatively easy step using just primers and PCR. See here for details.

Reporter

Glycogen Synthase Kinase 3 β (Fig) is known to be phosphorylated by several enzymes in eukaryotic cell. We used first 13 amino acid sequence to construct a reporter (GSK Reporter) which phosphorylation state could be detected. 9th amino acid which serine is phosphorylated in eukaryotic cell(Fig). 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.

GSK tag was constructed by Julie Torruellas Garcia, Gregory V. Plano et al. We removed present Spe1 site in the sequence by silent mutation

Translation: M   S   G   R   P   R   T   T   S-p  F   A   E   S
Original   :ATG AGT GGT CGC CCT CGC ACT ACT  AGT TTC GCT GAA AGT
rm SPE1    :ATG AGT GGT CGC CCT CGC ACT ACA* AGT TTC GCT GAA AGT 

Phosphorylated Serine is shown as S-p.

GSK tag can be added to N terminus, C terminus*1 and anywhere in between*2 of the protein. We opted to insert it between SlrP secretion tag and the protein we wonted to inject. To SlrP is required to be on CorN terminus. And insertin on C trminus would have required some costly primers.

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]
2. JWensheng Luo and Michael S. Donnenberg. 2011. Interactions and Predicted Host Membrane Topology of the Enteropathogenic Escherichia coli Translocator Protein EspB. J. Bacteriol.Vol.193:2972–80. [http://www.ncbi.nlm.nih.gov/pubmed/21498649 PubMed]