Team:Macquarie Australia/Results

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Results and Characterisation

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Results

Heme Oxygenase

Bacteriophytochromes

Sequencing Data

Heme Oxygenase

Bacteriophytochromes

Characterisation

Heme Oxygenase

Bacteriophytochromes

The Switch


Heme Oxygenase Results

We produced a Heme Oxygenase BioBrick 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). We used a 1kb ladder.

Heme Oxygenase Sequencing Results

The plasmid was sequencing using the forward and reverse primers for the BioBricks. We performed Blastx pipeline to determine if there was a significant change in the protein sequence.

SampleProposed Identity e-valueMaxID
1C-6FHeme Oxygenase (Synechocystic sp. PCC603)7e-17699%
1C-6RHeme Oxygenase (Synechocystic sp. PCC603)1e-17199%
1C-4FHeme Oxygenase (Synechocystic sp. PCC603)4e-17699%
1C-4RHeme Oxygenase (Synechocystic sp. PCC603)6e-17299%
1C-5FHeme Oxygenase (Synechocystic sp. PCC603)1e-2392%
1C-5FHeme Oxygenase (Synechocystic sp. PCC603)6e-17299%

Given that this was the source of our gene, we proposed that the sequencing result was accurate. We then compared to the original gBlock sequence and determined that the sequencing was accurate and confirmed the identity of the plasmid.


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 incubated 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. We observed this as well in our assembled switch. The image below demonstrates the green produced compared to uninduced Heme Oxygenase and the Bacteriophytochrome.


Bacteriophytochromes Results

Like the Heme Oxygenase, the bacteriophytochromes from Deinococcus radiodurans and Agrobacterium tumefaciens were optimised for use in E. Coli. The identity of the plasmid was determined by sequencing and by digestion.


Bacteriophytochrome Sequencing

SequenceProposed IdentityE valueMax ID
F3CE1Chain A, Crystal Structure Of A Monomeric Infrared Fluorescent Deinococcus Radiodurans Bacteriophytochrome Chromophore Binding Domain3.00E-16099%
R3CE1photoreceptor [Deinococcus radiodurans R1] Full=Bacteriophytochrome9.00E-65 85%
F3CE2Chain A, Crystal Structure Of A Monomeric Infrared Fluorescent Deinococcus Radiodurans Bacteriophytochrome Chromophore Binding Domain2.00E-16199%
F3CE3Chain A, Crystal Structure Of A Monomeric Infrared Fluorescent Deinococcus Radiodurans Bacteriophytochrome Chromophore Binding Domain8.00E-16399%
F4KE1Agp1-AGP2 fusion protein [synthetic construct]0.00E+0099%
F4KE2Agp1-AGP2 fusion protein [synthetic construct]099%
R4KE2bacteriophytochrome protein [Agrobacterium tumefaciens str. C58]099%
F4Ke3Agp1-AGP2 fusion protein [synthetic construct]0100%
R4KE3phytochrome Agp1 [synthetic construct]095%
F5CE1Agp1-AGP2 fusion protein [synthetic construct]0100%
R5CE1bacteriophytochrome protein [Agrobacterium tumefaciens str. C58]099%
F5CE2Agp1-AGP2 fusion protein [synthetic construct]099%
R5CE2phytochrome Agp1 [synthetic construct]0.00E+0098%
F5CE3phytochrome Agp1 [synthetic construct]4.00E-11383%
R5CE3bacteriophytochrome protein [Agrobacterium tumefaciens str. C58]099%

The Blastx pipeline suggested that there was a near identical match to the orginal source for almost all of the sequencing performed. Blastn were run to determine the deviance from the theoretical sample.


Bacteriophytochrome Characterisation


The Switch

Two of the BioBricks produced were ligated together to produce the light switch. We demonstrated that the switch was produced by inspecting a gel following ligation and then digestion. The gel can be seen below.

Gel 1: We have run against a Heme Oxygenase standard (Lane 1). The gel contains digested fragments from our composite BioBrick (Heme Oxygenase and Agro). The upper band (Black Box) is the Heme Oxygenase with the bacteriophytochrome and the bottom band (blue) is the plasmid backbone.

This provided the evidence that the product had been successfully ligated.

An SDS page gel was run of the ligation products to observe if the heme oxygenase was able to produce biliverdin. The biliverdin binds to a specific site in the bacteriophytochrome. As biliverdin is IR active this coupling can be ovbserved under infrared (IR) light.