Team:BCCS-Bristol/Wetlab/K381001 Construction

From 2010.igem.org

(Difference between revisions)
m (New page: Having decided on our biobrick design, our first task was to aquire the necessary parts and construct our first biobrick. BBa_I13522 was taken from well 8A kitplate 2 of the 2010 distribu...)
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Construction of the biobrick can be broadly broken down into
Construction of the biobrick can be broadly broken down into
-
*Generating larger quantities of both our component biobricks
+
* Generating larger quantities of both our component biobricks
-
*Digesting each of these biobricks
+
* Digesting each of these biobricks
-
*Ligating together the desired parts to create a new biobrick
+
* Ligating together the desired parts to create a new biobrick
-
*Generating larger quantities of this biobrick, checking and sequencing
+
* Generating larger quantities of this biobrick, checking and sequencing
 +
 
 +
 
 +
Generating large quantities of DNA:
 +
* Whilst waiting for DNA from the parts registry we tried to obtain large amounts of the constitutive GFP biobrick
 +
* We initially tried transforming MG1655s with DNA from the kit plate but were unsuccessful. Suspected the problem was down to cells not being competent enough and insufficient DNA in kit plate.
 +
* Thus we tried transforming two further strains; XL1 Blues and commercial NovaBlues (see table for details)
 +
 
 +
{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000"
 +
!style="border-style: solid; border-width: 1px"| XL1-blue positive control
 +
!style="border-style: solid; border-width: 1px"| 100μL cells + 2μL known plasmid solution
 +
|-
 +
!style="border-style: solid; border-width: 1px"| XL1-blue negative control
 +
!style="border-style: solid; border-width: 1px"| 100μL cells (untransformed)
 +
|-
 +
!style="border-style: solid; border-width: 1px"| XL1-blue + 2009 biobrick
 +
!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2009 biobrick
 +
|-
 +
!style="border-style: solid; border-width: 1px"| XL1-blue + 2010 biobrick
 +
!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2010 biobrick
 +
|-
 +
!style="border-style: solid; border-width: 1px"| Nova blue + 2009 biobrick
 +
!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2009 biobrick
 +
|-
 +
!style="border-style: solid; border-width: 1px"| Nova blue + 2010 biobrick
 +
!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2010 biobrick
 +
|}
 +
 
 +
* Only the commercial cells were successful and provided us with colonies
 +
* Colonies then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added)
 +
* These overnights were then miniprepped using QIAGEN kit to obtain 50μL of concentrated DNA

Revision as of 11:16, 17 September 2010

Having decided on our biobrick design, our first task was to aquire the necessary parts and construct our first biobrick.

BBa_I13522 was taken from well 8A kitplate 2 of the 2010 distribution BBa_K216005 was requested from the parts registry

Construction of the biobrick can be broadly broken down into

  • Generating larger quantities of both our component biobricks
  • Digesting each of these biobricks
  • Ligating together the desired parts to create a new biobrick
  • Generating larger quantities of this biobrick, checking and sequencing


Generating large quantities of DNA:

  • Whilst waiting for DNA from the parts registry we tried to obtain large amounts of the constitutive GFP biobrick
  • We initially tried transforming MG1655s with DNA from the kit plate but were unsuccessful. Suspected the problem was down to cells not being competent enough and insufficient DNA in kit plate.
  • Thus we tried transforming two further strains; XL1 Blues and commercial NovaBlues (see table for details)
XL1-blue positive control 100μL cells + 2μL known plasmid solution
XL1-blue negative control 100μL cells (untransformed)
XL1-blue + 2009 biobrick 100μL cells + 5μL 2009 biobrick
XL1-blue + 2010 biobrick 100μL cells + 5μL 2010 biobrick
Nova blue + 2009 biobrick 100μL cells + 5μL 2009 biobrick
Nova blue + 2010 biobrick 100μL cells + 5μL 2010 biobrick
  • Only the commercial cells were successful and provided us with colonies
  • Colonies then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added)
  • These overnights were then miniprepped using QIAGEN kit to obtain 50μL of concentrated DNA
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