Team:CIDEB-UANL Mexico/project union

After E.CARU performs its other tasks, in order to remove E. coli from the salty water for making it useful, the ability for binding to silica was introduced to the bacteria. Using this ability, it would be easy to clean the water free of bacterium through a biological filter.

Description

L2

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

AIDA

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).

How do L2 and AIDA act together?

As AIDA is a membrane protein producer and L2 produces a protein for making possible the attachment to silica, the team decided to make a fusion protein, so that the protein produced by L2 can be anchored to the membrane of the bacteria with the help of AIDA, and in that way, binding the E.CARU to silica pearls for being filtered.

Why is it important to use BgIII and BamHI to link L2 and AIDA-I?

It was needed to join both proteins in order to make a fusion protein, but SpeI and XbaI could not be used to join them because the reading frame would change, coding for a completely different protein. In order to avoid such problem, BgIII and BamHI were used instead, which could join AIDA and L2 without changing the reading frame. The scar produced between BamHI and BgIII, as it is shown in Figure 3, is formed by six bases, respecting the reading frame from both proteins in order to synthetize the correct protein.

Device

Initially, L2+AIDA and IrrE, protein for giving resistance to E. coli to adverse conditions (Resistance module), were joined together in only one circuit, but we needed to separate them because L2+AIDA has not been tested and it could affect the production of IrrE (see Figure 4.) as well as for testing each module alone.