Team:UC Berkeley/Assembly

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Revision as of 01:48, 29 October 2008

Assembly

Our goal is to simplify two antibiotic assembly eliminating the replace mini-prep steps and making the protocol reagent-free. This will be accomplished by using our lysis device to lyse cells and extract DNA and engineering cells to produce their own restriction enzymes and ligase.

For a general overview of two antibiotic assembly, click here [[1]]

Assembly in Cell Lysate

Our lab currently using cells that are engineered to methylate either BamHI or BglII restriction sites. Part A is transformed into a "lefty" cell that is methylated on BglII restriction sites while Part B is transformed into a "righty" cell that is methylated on BamHI restriction sites.

We propose to integrate the BamHI and BglII genes into the E. coli genome. In this scheme, lefty cells that are methylated on BglII cut sites, will stably express ligase and BglII. Righty cells that are methylated on BamHI cut sites, will be engineered to stably express XhoI and BamHI. Since restriction enzymes will not cut methylated DNA, the BglII restriction site in the lefty cell and the BamHI restriction site in the righty cell are blocked from digestion. The methylation also protects the cellular DNA from being cut when these genes are expressed.

In a single eppendorf tube, lefty and righty cells containing the plasmids described above are mixed and pelleted. The supernatant is discarded and the cells are resuspended in NEB2. The cells are then lysed using our lysis device. Click here for more information on our lysis device. [[2]].

The buffered lysate will contain all of the enzymes necessary for two antibiotic assembly. The lysate is incubated for one hour at 37 degrees and 30 minutes at 25 degrees. 10uL of lysate is transformed into new cells and the cells screened by plated on the proper antibiotic and growing overnight.

We have begun testing of our BamHI and ligase strains of E. coli by using commercially available restriction enzymes and ligase.

Assembly in-Vivo

We propose to engineer cells that are methylated on BamHI and BglII cut sites and stably express BamHI, BglII, Cre and ligase. These cells will be infected with two phagemids, each containing a basic part.

@@ Line 2: / Line 2: @@
 <div style="text-align: center;"><font size="6">'''Assembly'''</font></div><br>
-The six assembly plasmids contain two of three different antibiotic resistance markers (kanamycin, ampilicin and Chloramphenicol) separated by a XhoI restriction site. The basic parts are flanked by BglII and BamHI restriction sites
+{{UCBmain}}
+<div style="text-align: center;"><font size="6">'''Assembly'''</font></div><br>
-[[Image:assemblyplasmid.jpg]]
+Our goal is to simplify two antibiotic assembly eliminating the replace mini-prep steps and making the protocol reagent-free. This will be accomplished by using our lysis device to lyse cells and extract DNA and engineering cells to produce their own restriction enzymes and ligase.
-The choice of antibiotic resistance markers in the assembly plasmids is predetermined such that once the two plasmids recombine, the new plasmid will have a combination of markers from the two assembly vectors. Although there are four possible products of this reaction, only one will have both of the correct antibiotic resistance markers.
+For a general overview of two antibiotic assembly, click here [[https://2008.igem.org/Team:UC_Berkeley/LayeredAssembly]]
-[[Image:cdb2ab.jpg]]
+==''Assembly in Cell Lysate''==
-The assembly plasmids are transformed into cells that are engineered to either methylate BamHI or BglII restriction sites. Part A is transformed into a "lefty" cell that is methylated on BglII restriction sites while Part B is transformed into a "righty" cell that is methylated on BamHI restriction sites.
+Our lab currently using cells that are engineered to methylate either BamHI or BglII restriction sites. Part A is transformed into a "lefty" cell that is methylated on BglII restriction sites while Part B is transformed into a "righty" cell that is methylated on BamHI restriction sites.
-Lefty cells are engineered to stably express ligase and the BglII restrictions enzyme and are methylated on BglII cut sites. Righty cells are engineered to stably express XhoI and BamHI restriction enzymes and are methylated on BamHI cut sites. These cells are co-transformed with a plasmid containing our lysis device.
+We propose to integrate the BamHI and BglII genes into the ''E. coli'' genome. In this scheme, lefty cells that are methylated on BglII cut sites, will stably express ligase and BglII. Righty cells that are methylated on BamHI cut sites, will be engineered to stably express XhoI and BamHI. Since restriction enzymes will not cut methylated DNA, the BglII restriction site in the lefty cell and the BamHI restriction site in the righty cell are blocked from digestion. The methylation also protects the cellular DNA from being cut when these genes are expressed.
-In a single eppendorf tube, lefty and righty cells containing basic part DNA are lysed using our lysis device. Click here for more information.[[https://2008.igem.org/Team:UC_Berkeley/LysisDevice]]
+In a single eppendorf tube, lefty and righty cells containing the plasmids described above are mixed and pelleted. The supernatant is discarded and the cells are resuspended in NEB2. The cells are then lysed using our lysis device. Click here for more information on our lysis device. [[https://2008.igem.org/Team:UC_Berkeley/LysisDevice]].
+The buffered lysate will contain all of the enzymes necessary for two antibiotic assembly. The lysate is incubated for  one hour at 37 degrees and 30 minutes at 25 degrees. 10uL of lysate is transformed into new cells and the cells screened by plated on the proper antibiotic and growing overnight.
-The basic part DNA, along with the restriction enzymes are released into solution. Since restriction enzymes will not cut methylated DNA, the restriction site BglII site in the lefty cell and the BamHI restriction site in the righty cell are blocked from digestion. The BamHI site in lefty and the BglII site in lefty, and the Xho sites in both plasmids will be cut. Ligase will join the xho sites and the BglII/BamHI complimentary sticky ends.
+[[Image:cdb6.jpg]]
-The assembly reaction can be repeated to combine a number of basic parts in a desired order.
+We have begun testing of our BamHI and ligase strains of ''E. coli'' by using commercially available restriction enzymes and ligase.
-Once the composite part is complete, it is transferred to a destination plasmid.
+==''Assembly in-Vivo''==
+We propose to engineer cells that are methylated on BamHI and BglII cut sites and stably express BamHI, BglII, Cre and ligase. These cells will be infected with two phagemids, each containing a basic part.