Team:CIDEB-UANL Mexico/project union
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<p>The gene L2 encodes for a protein able to attach to silica. Taniguchi et al. reported in 2007 that the L2 ribosomal protein from <i>E. coli</i> 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.</p> | <p>The gene L2 encodes for a protein able to attach to silica. Taniguchi et al. reported in 2007 that the L2 ribosomal protein from <i>E. coli</i> 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.</p> | ||
- | <center><p><img width=237 height= | + | <center><p><img width=237 height=180 src="https://static.igem.org/mediawiki/2014hs/0/05/Silica_washed.png" |
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<p>AIDA-I is an <i>E. coli</i> 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.</p> | <p>AIDA-I is an <i>E. coli</i> 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.</p> | ||
- | <center><p><img width=226 height= | + | <center><p><img width=226 height=251 src="https://static.igem.org/mediawiki/2014hs/a/a2/Aida_system.png" |
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<p>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 2, is formed by six bases respecting the reading frame from both proteins in order to synthetize the correct protein.</p><br> | <p>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 2, is formed by six bases respecting the reading frame from both proteins in order to synthetize the correct protein.</p><br> | ||
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<p>We will use a silica biofilter to remove <i>E. coli</i> from water, but in order to observe if really <i>E. coli</i> 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 <i>E. coli</i> with two plasmids, one with RFP and the other containing the fusion protein (<b>figure 6</b>); if the biofilter becomes red it would mean <i>E. coli</i> is attached to it.</p> | <p>We will use a silica biofilter to remove <i>E. coli</i> from water, but in order to observe if really <i>E. coli</i> 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 <i>E. coli</i> with two plasmids, one with RFP and the other containing the fusion protein (<b>figure 6</b>); if the biofilter becomes red it would mean <i>E. coli</i> is attached to it.</p> | ||
- | <center><p><img width=401 height= | + | <center><p><img width=401 height=243 src="https://static.igem.org/mediawiki/2014hs/c/c3/E.COLI_RFP.png" |
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Revision as of 21:22, 14 June 2014
Union Module
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 E. coli 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. |
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How is the Union module composed?
The binding 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.
Figure 2. Proteins absorbed to a silica slide and washed for 24 hours a)GFP b) L2-GFP fusion c) R9-GFP fusion. Taken from Taniguchi (2007)
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.
Figure 3. Schematic representation of AIDA-I carrier protein
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 2, is formed by six bases respecting the reading frame from both proteins in order to synthetize the correct protein.
Figure 4. Example of a ligation using BamHI and BgIII
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 6); if the biofilter becomes red it would mean E. coli is attached to it.
Figure 5. E. coli containing the fusion (L2+AIDA) and RFP proteins
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
IMAGE |
CODE |
DESCRIPTION |
|
The J23119 is the most effective
and common constitutive promoter used. It has a length of 35bp. |
|
|
This specific RBS is based on Elowitz repressilator. It
has a length of 12bp. |
|
|
L2. This CDS gives the property
for binding silica and glass surfaces to E. coli, it has a length of 819 bp. |
|
|
AIDA-I is synthetized as a 132 kDa pre-protein featuring
a signal peptide which is cleaved during transport trough the inner membrane,
a 78 kDa adhesin (passenger) domain, and a 45 kDa translocator. This
autotransporter has a large capability in translocating relatively large
passengers from 12-65 kDa by showing a N-terminal type of fusion. Coupled with
a passenger domain and a signal peptide (K888005), it is possible to express
functional proteins in the outer membrane of E. coli. It has a length of 1482 bp. |
|
|
When this part is coupled with a
passenger attached to AIDA-I translocator domain (K888001), it is possible to
express functional proteins in the outer membrane of E. coli. The signal
peptide is naturally cleaved during transport trough the inner membrane (Li
et al. 2007; van Bloois et al. 2011).It has a length of 147 bp. |
|
|
Part made of 6bp, responsible for stopping
transcription. |
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.
References
● Antiquity (2003). Part:BBa_B0034. Retrieved on March 30th, 2014, from: http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034.
● iGEM2006_Berkeley (2006). Part:BBa_J23119. Retrieved on April 30, 2014, from: http://parts.igem.org/wiki/index.php?title=Part:BBa_J23119.
● iGEM12_UANL_Mty-Mexico (2012). Part BBa_K888000. Retrieved on March 29th, 2014, from: http://parts.igem.org/wiki/index.php?title=Part:BBa_K888000.
● iGEM12_UANL_Mty-Mexico (2012). Part BBa_K888001. Retrieved on March 29th, 2014, from: http://parts.igem.org/wiki/index.php?title=Part:BBa_K888001.
● iGEM12_UANL_Mty-Mexico (2012) Part BBa_K888005. Retrieved on March 29th, from: http://parts.igem.org/wiki/index.php?title=Part:BBa_K888005.
● UANL Mexico (2012). Recovery module. Retrieved on March 28th, from: https://2012.igem.org/Team:UANL_Mty-Mexico/Project/recovery.