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 translocated 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.

The union circuit consists mainly of a constitutive promoter, an RBS, a peptide signal attached to L2 and this attached to AIDA by a scar and a terminator. The device is designed this way in order to produce a protein that helps the bacteria to bind to silica.

Parts of the module

Justifications

Although E. coli could acquire the ability to bind to silica, a biological filter was designed to remove bacteria from water. The team´s proposed biofilter is shown next:

Silica was chosen as the ideal material because it is cheap and it is commonly found and also because the team wanted to give this material a new use. It was also chosen in order to finish the UANL Mexico 2012 team work. They created a circuit to make E. coli bind to silica, but as they did not prove it, we wanted to determine if it is functional.

AIDA-1 allows the expression of proteins larger than small peptides in the outer membrane, which is why it is the best option to use with L2. AIDA-I was obtained by PCR by UANL Mexico 2012, so we decided use their piece for our project, as it was easy for the team to obtain it because both of the teams are in the same city.

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. However, their project was not finished.

Union Module Zoom In

Bibliography/References