Team:CIDEB-UANL Mexico/project union

E. coli needs to resist saline environments, UV rays and temperature changes in order to capture Na+ ions, and produce an aroma as a reporter, everything in the water. But after E. coli performs its tasks, it is necessary to remove it from the water in order to obtain usable water; it was easy to do it through a biofilter. We chose silica as the material for our biofilter, so that E. coli expressed a membrane protein which could have the ability for binding silica, and in that way remove E. coli from the water. This was possible for the circuit created by UANL Mexico 2012 team; they created a circuit to make E. coli attached to silica, but as they did not proved it, we want to determine if it really works or not.

How is the Union module composed?

Initially, L2+AIDA and IrrE, protein for giving resistance to E. coli to adverse conditions (resistance module [link]), were joined together in only one circuit, but we needed to separate them because L2+AIDA has not been proved yet and it could affect the correct production of IrrE (see figure 1) as well as for testing each module alone.

The union circuit consists mainly in a fusion protein (a set which includes the CDS L2 with its peptide signal and AIDA) in order to make the protein for binding silica, a membrane protein. In that way E. coli would attach to silica.

How does L2 and AIDA act together?

The gene L2 encodes for a protein able to attach to silica. Taniguchi et al. reported in 2007 that the L2 ribosomal protein from E. coli strongly adsorbs to silica surfaces, up to 200 times tighter than poliarginine tags commonly used for protein purification. In their work, Taniguchiet al. 2007, constructed a fusion protein of L2 and green fluorescent protein (GFP) which adsorbed to a silica surface even after washing for 24 hours with a buffer containing 1 M NaCl (Figure 3). UANL Mexico 2012 did not have this piece in stock, so we decided to synthetize L2.

AIDA-I is an E. coli membrane protein with a passenger domain of 76 kDa exposed to the extracellular space and a transmembrane beta-barrel domain of 45 kDa; the latter has been used to express functional proteins in the cell-membrane of up to 65 kDa (van Bloois et al., 2011). Furthermore passengers coupled to AIDA-I have been reported to reach an expression level of more than 100,000 copies per cell in the outer membrane (Jose and Meyer, 2007). AIDA-1 allows the expression of proteins larger than small peptides in the outer membrane what makes it the best option to use with L2. AIDA-I was obtained by PCR for UANL Mexico 2012, so we use their piece for our project.

How important to use BgIII and BamHI to link L2 and AIDA-I?

As we need to join both proteins in order to make a fusion protein we cannot use SpeI and XbaI to join them because the reading frame would change making a completely different protein. So in order to avoid such problem we use BgIII and BamHI instead which can join AIDA and L2 without changing the reading frame. The scar produced between BamHI and BgIII, as is shown in the figure 3, is formed by six bases respecting the reading frame from both proteins in order to synthetize the correct protein.

How to know if E.coli binds to silica?

We will use a silica biofilter to remove E. coli from water, but in order to observe if really E. coli would attach to it we wanted to use a Wintergreen aroma as reporter. This would lead us know if the bacteria are in the biofilter by adding salicylic acid and changing the temperature; but as we will test this module alone we needed to design a new way to observe if L2+AIDA works. We decided to transform E. coli with two plasmids, one with RFP and the other containing the fusion protein (figure 7); if the biofilter becomes red it would mean E. coli is attached to it.

How to create a biofilter?

Although E. coli could acquire the ability for binding silica, we need to create a biofilter to remove bacteria from water. Our proposal as biofilter is shown in the next figure:

Parts of the module

Other teams that used it:

UANL México 2012: They proposed the fusion protein for using it to binding silica after detect and capture arsenic acid in groundwater, and in that way removed the pollutant arsenic acid from the water, as part of water bioremediation, but they did not finish it. That is why we want to determine if it will work.

Union Module Zoom In

Bibliography/References