Abstract

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.

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 to see if they can be secreted and calculate their secretion speed.

Intro

Bacteria living around us evolved ways to effect their surrounding environment. This is done by using secretion systems. There are six known types of secretion systems: one through six. These are called Type One Secretion System (T1SS), T2SS, T3SS and so on. Some system only secrete designated proteins outside, others can translocate proteins directly to other cells. Secretion systems able to inject proteins into other cell caught imagination of many researchers.

Ours was no exception. We chose to use T3SS for our experiments. During iGEM 2010 we found that E.Coli with a part of Salmonella genome library expresses T3SS. This presented opportunity to work with the amazing machinery without involving pathogenic bacteria.

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 continued to characterise it. An further develop data sheet of T3SS possibilities.

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. 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. It 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 tag, Bsa I Cloning Site. Desired protein can be 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 tag would have been a laborious task which we didn't want to endure. So ready-to-inject backbones was proposed(Fig.1).

We designed Bsa I 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. This property is exhibited by first 13aa of this protein^[1] which makes it very small tag. Thus we now can easily detect if protein was injected in eucaryotic cells. Because of it's small size the interference on tag protein should be at minimum.

See here for details.

We combining these futures in a backbone and constructed ready-to-inject bakcbone. SlrP is an injection signal, without it the protein cannot be secreted. GSK is a tag, by detecting phosphorylation of it you can distinguish whether it has been it eukaryotic cell. For us it is an evidence of successful injection. Bsa I 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.

We used ready-to-inject backbone to inject five proteins representing different structures and functions to see which get injected. This was are shot to try and characterise T3SS a little bit further.

See here for details.

---brief summary of the injection results---

Bsa I Cloning Site

Bsa I 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. Bsa I cloning site is valuable part when you need change particular part in the middle of the construct. It was designed that inserted biobrick would be fused to preceding signals.

Bsa I restriction enzyme is in distinguish group of enzyme which cutting site is different from recognition site. Unlike EcoR I or Pst I, 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). Which is the key future making insertion in the middle of construct possible.

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 EcoR I digested strand. As 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 Bsa I. You cannot use more than one Bsa I cloning site per construct. However, using other enzymes of this kind it is possible to add additional insertion sites per plasmid.

For our construct we designed a cloning site which when digested with Bsa I will produce Not I like overhang and Spe I like overhang (Fig). Which will ligate to Not I and Spe I but won't be digested after.

         Bsa I    Not I'           Spe I'   Bsa I
  
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'

However there are some limitations Bsa I. Its not an official biobrick restriction enzyme so you have to screen each whole construct for Bsa I recognition sequences. However no worries are needed for inserts. Because only official restriction enzymes treatment is required for them.

Usage standard assembly produces in-frame stop codons in scars. We got around this by using PCR to amplify our inserts. We designed amplification primers to insert mutation and remove both remove change stop codon and Xba I restriction site.

GSK tag

Glycogen Synthase Kinase 3β is known to be phosphorylated by several enzymes in eukaryotic cell. We used first 13 amino acid as a tag (GSK tag)^[1]. Ninth amino acid, serine is phosphorylated in eukaryotic cell(Fig). This phosphorylation state could be detected by using phsopho-specific antibodies which bind to only phosphorylated GSK tag. This way it is possible to distinguish whether GSK tag 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 Spe I 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 Spe I   :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^[1], C terminus^[1] and anywhere in middle^[2] of the protein. We opted to insert it between SlrP secretion tag and the protein we wanted to inject.

Using non-phosphospecific antibodies it is possible to check the total amount of expressed protein with the tag. Comparing it with the injected protein you can determine the efficiency of the injection.

By comparing the mass of the protein with GSK tag it is also possible to see if it had been modified in eucaryotic cell. It can be used alongside of TEV site and provide proof for successful TEV protease activity. An experiment we would like to try in the future.

Investigation of T3SS-injectable proteins

Here we will discuss the structure of proteins which are injected and which are not. We tried five different proteins: mnt repressor, RFP, GFP, Cre DNA recombinase, (CCR5) transmembrane, LacI and Luciferase. All were chosen from biobrick distribution. As these parts are widely used in iGEM studying them would have a bigger impact compared to exotic ones.

Our main concern was not with the size but the stability of proteins. Previous research show that proteins like Zinc-Fingers are very stable and couldn't be injected. Hight stability prevents unfolding by T3SS chaperons.

We showed that GFP can be injected into eucaryotic cells by confocal laser microscope imaging. Thus it can serve as a control. Next is RFG, a fluorescent protein but with different structure from GFP.

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]