Team:ITESM Mexico/Experimental design

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{{Team:ITESM_Mexico/Top}}
{{Team:ITESM_Mexico/Top}}
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::''Concentration and photoreceptor''
 
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:::::'''Basic steps:'''
 
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::*Transformation of competent cells with iGEM parts and our own designed parts
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<html><h1 class="myowntitle">Experimental design</h1> </html>
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------
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::'''Concentration and photoreceptor'''
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::*Plasmid isolation of each part
 
-
::*Run an electrophoresis gel to detect the presence of DNA
+
::''Basic steps:''
-
::*Purification of gel bands to get the parts of interest
+
*'''Transformation of competent cells with iGEM parts and our own designed parts '''
 +
For both constructs parts need to be transformed. The best strain to perform the transformation is with DH5-α. The transformation is performed by the kit TransformAid Bacterial Transformation kit from Fermentas. The parts must be transformed, cultured and subcultured. There are parts with different resistances; two of the antibiotics were dissolved in water (ampicillin and kanamycin), and chloramphenicol was dissolved in absolute ethanol.  The transformation is performed with synthesized parts from Genscript and the plates sent by IGEM. Then the parts are grown in bacteria by incubating them at 37°C overnight.
-
::*Digestion of the pieces with EcoRI, XbaI, SpeI, PstI enzymes
+
*'''Plasmid isolation of each part '''
 +
After the transformation of bacteria the parts have to be taken to miniprep, the minipreps are proven to be better when the plasmid isolation is performed by the kit PureYield Plasmid Miniprep System from Promea, which is better than the manual method of alkaline lysis with “home-made” solutions. This kit improves the quantity of DNA obtained. This data can be compared with spectrophotometer tests by comparing the absorbance at 280 nm and 260 nm.
 +
*'''Digestion of the pieces with EcoRI, XbaI, SpeI, PstI enzymes'''
 +
After the quantity of DNA is determined digestion has to be made, to obtain the pieces of interest, thus the ligation can take place and the construct can be built. For this assembly we decided to follow a parallel assembly where several parts are cleaved with two enzymes and the other part with two enzymes to obtain the parts desired without complications.
 +
There are pieces that are only cut by EcoRI and SpeI, and there are other parts that are cut by EcoRI and Xbal. But for the final assembly and the assembly of the individual pieces, the backbone, which has resistance to chloramphenicol, is cut by EcoRI and PstI.
 +
Digestion is followed by the kit Biobrick Assembly from New England
 +
*'''Run an electrophoresis gel to detect the presence of DNA'''
 +
[[File:Experimental.jpg|center]]
 +
After digestion an electrophoresis gel must be run. There are pieces from 35 bp to 3 kb in the construct; therefore gel must be run at different concentrations of agarose. Also we found a method that helps us to run gels 20 times faster than normal gels. This by applying lithium acetate and lithium buffer.
 +
*'''Purification of gel bands to get the parts of interest'''
 +
According to the kit from New England there is no need of Gel electrophoresis after digestion but is also recommended to do it to assure the presence of the parts by the presence of a band in a gel. If the gel is run a purification must be performed to obtain the DNA from the bands
 +
*'''Ligation of the pieces'''
 +
Pieces are ligated with the kit Biobrick Assembly from New England.  Then a gel has to be performed to obtain the DNA. And after  that gel is obtained a new purification and digestion can be done or a transformation in new bacteria.
 +
*'''Repeat of the process of digestion and ligation to put together the whole construct '''
-
::*Run electrophoresis gel to detect the digestion products
+
*'''Confirmatory test'''
-
 
+
To confirm the correct functioning of the construct tests with fluorocytometer must be performed, to quantify the expression when all the parts are activated or just some of the parts.
-
::*Purification of the pieces from the gel
+
-
 
+
-
::*Ligation of the pieces
+
-
 
+
-
::*Repeat of the process of digestion and ligation to put together the whole construct
+
-
 
+
-
::*Confirmatory test
+
-
 
+
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:::::'''Experimental Dessign:'''
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::Photoreceptor mechanism was used from the the Tokio-Nokogen team of 2009 (https://2009.igem.org/Team:Tokyo-Nokogen) This mechanism was modified by adding only the green light receptor instead of the whole mechanism., including also the red light receptor mechanism.
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::The most important modification of this mechanism was the inclusion of the protein RecA in our construct to make it compatible to the regulation system of lambda phage incorporated in the mechanism of concentration.
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::Green receptor: Is going to be used to initiate the entire mechanism. It is composed by eight parts, in a sequence of twelve parts. 
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::*The constitutive promoter is used for the permanent expression of the gene, it is attached to a ribosome binding site (RBS) that is a sequence on the messenger RNA to which the ribosome can bind and initiate protein translation and produce the ho1 (heme oxigenase 1) that converts heme to biliverdin IXalpha.
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::*The sequence that produce pcy4A (phycocyanobilinàferredoxin oxidoreductase) allows the convertion of the biliverdin IXalpha to phycocyanobilin.
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::*Another RBS is used to produce CcaS-EnvZ which is a chimeric protein of ScaS and the EnvZ that acts as the light responsive domain. CcaS-EnvZ is the coding sequence of the green light receptor, it induces the expression of phycobilisome linker protein.
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::*The green light OmpR regulates the production of outer membrane proteins. This  promoter will allow the expression of the RecA protein that activates the expression of the promoters for arabinose concentration by reducing the activity of the lambda repressor.
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::*RecA is a protein used for the cleavage of protein lambda. It has shown that it has a cleavage activity when its binded to lambda repressor. This is a essential part of the project, because these interactions are the link between the photoreceptor and the concentration mechanims by the lambda repressor and the lambda operators. (Sauer et al, 1981)
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-
 
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:::::'''Concentration:'''
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::The concentration mechanism is based in the experiments and mechanisms developed by British Columbia University iGEM team in 2009 ( https://2009.igem.org/Team:British_Columbia).  This  is the part we focused on improving from the original idea. Basically the experiment was the same, there are only few changes from the original construct, like the addition of an anti-sense key to stop the action of the key that disables the capability of the reporter protein to express.
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::The construct is very similar to the one from UBC. We took the pieces Pbad st for sensing low concentrations and Pbad wk for sensing high concentrations. We re-designed the activation/inactivation (lock and key) based on the experiments of lock and key developed by UBC in 2009 and the control of gene expression (Isaacs et al,2004)
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::We re-designed some pieces to make them more specific, we modified the lock and key mechanism, and added more parts, one lock and key specific for the high concentration and other lock and key designed for low concentrations. Also the inclusion of one new part that regulates one of the keys by inactivating it.
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::The first part of the second construct consists in the constitutive promoter that produces the lambda repressor protein. This lambda repressor binds to the operators, inhibiting the expression of the mechanisms of concentration expression. The operators interacts with the repressor lambda inhibiting the action of the RNA polymerase to transcribe the sequence.
+
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::The next link is between the concentration mechanism and the lambda repressor. Those are the operators, that can be found before the promoter Pbad st. these operators are responsible for the binding of lambda repressor protein and for the expression of the reporter protein. If the operators are free then there can be expression. Operators can only be free once the RecA protein cleavages the lambda repressor, so the expression can be continued. Then the Pbad st can activate the expression of the reporter protein GFP, but there is a previous step where there is the first lock (crxst);  This first lock inhibits the expression of the reporter protein and that’s why at the same concentration with the same promoter there is the activation of our key (tast).
+
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For high concentrations there is the promoter pbad wk, which can sense big quantities of arabinose. The promoter is also activated by action of the operators. Once the promoter is activated the process of expression is continued until the CFP is expressed. To express this protein the lock (crxwk) must be inactivated by the key (tawk). To assure that only the high concentration mechanism is enabled there is the necessity to turn down the low concentration, this is achieved by expressing an antisense key (itast) that inhibits the production of the low concentration key (tast) thus the low concentration lock (crxst) will activate and will inhibit the expression of GFP.
+

Latest revision as of 03:15, 29 September 2011

ITESM MÉXICO

SensE.coli

Igem Itesm


Experimental design

Concentration and photoreceptor


Basic steps:
  • Transformation of competent cells with iGEM parts and our own designed parts

For both constructs parts need to be transformed. The best strain to perform the transformation is with DH5-α. The transformation is performed by the kit TransformAid Bacterial Transformation kit from Fermentas. The parts must be transformed, cultured and subcultured. There are parts with different resistances; two of the antibiotics were dissolved in water (ampicillin and kanamycin), and chloramphenicol was dissolved in absolute ethanol. The transformation is performed with synthesized parts from Genscript and the plates sent by IGEM. Then the parts are grown in bacteria by incubating them at 37°C overnight.

  • Plasmid isolation of each part

After the transformation of bacteria the parts have to be taken to miniprep, the minipreps are proven to be better when the plasmid isolation is performed by the kit PureYield Plasmid Miniprep System from Promea, which is better than the manual method of alkaline lysis with “home-made” solutions. This kit improves the quantity of DNA obtained. This data can be compared with spectrophotometer tests by comparing the absorbance at 280 nm and 260 nm.

  • Digestion of the pieces with EcoRI, XbaI, SpeI, PstI enzymes

After the quantity of DNA is determined digestion has to be made, to obtain the pieces of interest, thus the ligation can take place and the construct can be built. For this assembly we decided to follow a parallel assembly where several parts are cleaved with two enzymes and the other part with two enzymes to obtain the parts desired without complications. There are pieces that are only cut by EcoRI and SpeI, and there are other parts that are cut by EcoRI and Xbal. But for the final assembly and the assembly of the individual pieces, the backbone, which has resistance to chloramphenicol, is cut by EcoRI and PstI. Digestion is followed by the kit Biobrick Assembly from New England

  • Run an electrophoresis gel to detect the presence of DNA
Experimental.jpg

After digestion an electrophoresis gel must be run. There are pieces from 35 bp to 3 kb in the construct; therefore gel must be run at different concentrations of agarose. Also we found a method that helps us to run gels 20 times faster than normal gels. This by applying lithium acetate and lithium buffer.

  • Purification of gel bands to get the parts of interest

According to the kit from New England there is no need of Gel electrophoresis after digestion but is also recommended to do it to assure the presence of the parts by the presence of a band in a gel. If the gel is run a purification must be performed to obtain the DNA from the bands

  • Ligation of the pieces

Pieces are ligated with the kit Biobrick Assembly from New England. Then a gel has to be performed to obtain the DNA. And after that gel is obtained a new purification and digestion can be done or a transformation in new bacteria.

  • Repeat of the process of digestion and ligation to put together the whole construct
  • Confirmatory test

To confirm the correct functioning of the construct tests with fluorocytometer must be performed, to quantify the expression when all the parts are activated or just some of the parts.

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