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Team:Imperial College London/Project/Switch/Results
From 2011.igem.org
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| + | <h2>Chapter 1: Assembly strategy</h2> | ||
| + | <p>The assembly of this module shall be the most challenging out of all of them. Not only are we starting it the latest, but we will be using parts from the registry to assemble it. The first step of assembly will require us to place the anti-Holin from the BBa_K112808 biobrick under the J23100 promoter in BBa_K398500. In order to perform this step we will be using a PCR which will contain non-homologous regions. These non-homologous sequences will contain the insulator, RBS (ITR obtained from modelling) and 15bp overhangs that will allow us to assemble the PCR products of both the biobricks through the use of In-Fusion. The PCR step will be incredibly challenging. Once the parts are correctly inserted into the pSB1C3 vector we will be able to extract it and use biobrick assembly to insert it into the Crim plasmid. Once in the Crim plasmid, the gene must be integrated into the genome. Once this step is completed we can proceed to the transformation of these cells (any attempts at transformation before we have these cells will just result in cell lysis).</p> | ||
| - | The first step was to extract the required biobricks from the registry, as we would need them many times each during the assembly. The part numbers and their locations in the 2011 distribution were looked up and then the DNA extracted by dissolving it in distilled water. We used RBS-RFP and the Cell Lysis Cassette from Berkeley 2008. | + | <p>We will also require the use of the J23103 promoter (the RPU which we have obtained from modelling)which can be found in a BBa_J61002 vector. We also have ordered an oligo of the promoter to run in parallel. Once this has been inserted into a pSB1C3 plasmids, we can extract the Holin and Endolysin genes from the BBa_K112808 biobrick using primers that will contains a SpeI and PstI site for biobrick assembly. Once the J23103 is assembled with the Endolysin and Holind we can transform the E. coli that contain the anti-Holin gene in the genome.</p> |
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| + | <p>The first step was to extract the required biobricks from the registry, as we would need them many times each during the assembly. The part numbers and their locations in the 2011 distribution were looked up and then the DNA extracted by dissolving it in distilled water. We used RBS-RFP and the Cell Lysis Cassette from Berkeley 2008.</p> | ||
<p> | <p> | ||
| - | This DNA was used to transform competent 5-alpha cells. The cells were transformed according to the protocol on the main page and then transformants were selected for by growing on ampicillin plates. The colonies which grew were picked, grown for around 8 hours in LB broth at 37°C and then mini-prepped. The DNA obtained from the mini-prep was stored on ice overnight. | + | This DNA was used to transform competent 5-alpha cells. The cells were transformed according to the protocol on the main page and then transformants were selected for by growing on ampicillin plates. The colonies which grew were picked, grown for around 8 hours in LB broth at 37°C and then mini-prepped. The DNA obtained from the mini-prep was stored on ice overnight.</p> |
<p> | <p> | ||
The next day, the DNA was digested using EcoRI and PstI, individually and together. The resulting digests were mixed with loading buffer and then run on an agarose gel at 90V for an hour. | The next day, the DNA was digested using EcoRI and PstI, individually and together. The resulting digests were mixed with loading buffer and then run on an agarose gel at 90V for an hour. | ||
| - | <p> | + | </p> |
| + | </body> | ||
| + | </html> | ||
Revision as of 10:44, 17 August 2011
Chapter 1: Assembly strategy
The assembly of this module shall be the most challenging out of all of them. Not only are we starting it the latest, but we will be using parts from the registry to assemble it. The first step of assembly will require us to place the anti-Holin from the BBa_K112808 biobrick under the J23100 promoter in BBa_K398500. In order to perform this step we will be using a PCR which will contain non-homologous regions. These non-homologous sequences will contain the insulator, RBS (ITR obtained from modelling) and 15bp overhangs that will allow us to assemble the PCR products of both the biobricks through the use of In-Fusion. The PCR step will be incredibly challenging. Once the parts are correctly inserted into the pSB1C3 vector we will be able to extract it and use biobrick assembly to insert it into the Crim plasmid. Once in the Crim plasmid, the gene must be integrated into the genome. Once this step is completed we can proceed to the transformation of these cells (any attempts at transformation before we have these cells will just result in cell lysis).
We will also require the use of the J23103 promoter (the RPU which we have obtained from modelling)which can be found in a BBa_J61002 vector. We also have ordered an oligo of the promoter to run in parallel. Once this has been inserted into a pSB1C3 plasmids, we can extract the Holin and Endolysin genes from the BBa_K112808 biobrick using primers that will contains a SpeI and PstI site for biobrick assembly. Once the J23103 is assembled with the Endolysin and Holind we can transform the E. coli that contain the anti-Holin gene in the genome.
The first step was to extract the required biobricks from the registry, as we would need them many times each during the assembly. The part numbers and their locations in the 2011 distribution were looked up and then the DNA extracted by dissolving it in distilled water. We used RBS-RFP and the Cell Lysis Cassette from Berkeley 2008.
This DNA was used to transform competent 5-alpha cells. The cells were transformed according to the protocol on the main page and then transformants were selected for by growing on ampicillin plates. The colonies which grew were picked, grown for around 8 hours in LB broth at 37°C and then mini-prepped. The DNA obtained from the mini-prep was stored on ice overnight.
The next day, the DNA was digested using EcoRI and PstI, individually and together. The resulting digests were mixed with loading buffer and then run on an agarose gel at 90V for an hour.
