Team:Macquarie Australia/Results

From 2012.igem.org

(Difference between revisions)
Line 39: Line 39:
<center><a name="3"><h3>Bacteriophytochromes</h3></center>
<center><a name="3"><h3>Bacteriophytochromes</h3></center>
 +
<p>Like the Heme Oxygenase, the bacteriophytochromes from <i>Deinococcus radiodurans</i> and <i>Agrobacterium tumefaciens</i> were optimised for use in <i>E. Coli</i>.</p>
<hr>
<hr>
<center><a name="4"><h3>Bacteriophytochrome Characterisation</h3></center>
<center><a name="4"><h3>Bacteriophytochrome Characterisation</h3></center>

Revision as of 04:52, 19 September 2012



Results and Characterisation

To quickly skip to the section that you wish to read, click on the links below.

Results

Heme Oxygenase

Bacteriophytochrome

Characterisation

Heme Oxygenase

Bacteriophytochrome


Heme Oxygenase

We produced a Heme oxygenase that was codon optimize for E. coli. The Gibson assembly of the T7 promoter containing Heme Oxygenase was successful. The transformation was successful with numerous colonies grown using Chloramphenicol as the selecting agent. Six colonies were selected and then they were sequenced before digestion with EcoR1 and Spe1. The sequencing suggested that all of the colonies contained the plasmid with a Heme oxygenase identical to the original protein sequence. The gel containing the digested Heme Oxygenase bearing plasmid can be seen in Figure 1.

Figure 1: The restriction digest showing the linearised plasmid backbone (Black Box) and the heme oxygenese gene (Green Box)

Characterisation of Heme Oxygenase

The T7 bearing Heme Oxygenase produced was characterised to determine if it was functional. BL21 E. coli was transformed with the plasmid, selected for using chloramphenicol and a culture was inoculated. The culture was then induced with ALA (d-aminolevulinic acid) for the heme pathway and IPTG to promote protein production. They were incubate overnight and the cells were spun down. We observed a functional Heme Oxygenase and the cells appeared a vibrant green after induction by ALA and IPTG.


Bacteriophytochromes

Like the Heme Oxygenase, the bacteriophytochromes from Deinococcus radiodurans and Agrobacterium tumefaciens were optimised for use in E. Coli.


Bacteriophytochrome Characterisation