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From 2012.igem.org

Results and Characterisation

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Results
Heme Oxygenase	Bacteriophytochromes

Sequencing Results
Heme Oxygenase	Bacteriophytochromes

Characterisation
Heme Oxygenase	Bacteriophytochromes	The Switch

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Heme Oxygenase Results

We produced a Heme Oxygenase BioBrick that was codon optimized 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 showed 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 (in increasing Kbp: 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10).

The lower band is the T7 Heme Oxygenase BioBrick (771 bp) which was expected. Similarly, the plasmid backbone was visible at the expected length (2070 bp). The similarity in intensity of the bands also demonstrates that the assembly and the digest were successful. The sequencing results for the six plasmids above can be seen below.

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Heme Oxygenase Sequencing Results

The Gibson Assembly performed was transformed into 6 different cultures of competent cells. Three of theses were selected for sequencing using both forward and reverse primers. They were sequenced using the forward and reverse primers for the BioBricks. We performed Blastx and Blastn pipelines (available here) to determine if there was a significant change in the protein sequence and to determine the identity of the plasmid. The sequences below are named to show: that it is heme oxygenase (1C), which transformation (4,5,6), and whether it is the forward or reverse primer (F or R).

Sample	Identity	E-value	MaxID
1C-6F	Heme Oxygenase (Synechocystic sp. PCC603)	7e-176	99%
1C-6R	Heme Oxygenase (Synechocystic sp. PCC603)	1e-171	99%
1C-4F	Heme Oxygenase (Synechocystic sp. PCC603)	4e-176	99%
1C-4R	Heme Oxygenase (Synechocystic sp. PCC603)	6e-172	99%
1C-5F	Heme Oxygenase (Synechocystic sp. PCC603)	6e-172	99%

We successfully assembled the codon optimised Heme Oxygenase. The probability that it is not Heme Oxygenase is negligibly small. The MaxID score is not 100% due to sequence misreads producing small gaps in the sequence. The E-value signifies the possibility of matches based purely on chance. A score of 0 identifies no background noise and is expected to be an error-free match.

The source of our gene was identified in the sequencing result which showed that the sequencing was accurate. We then compared to the original gBlock sequence and determined that the sequencing was accurate and confirmed the identity of the plasmid. The Blastn pipeline indicated that there was no significant change from the theoretical sequence. With this data we would assume that the protein would be functional and performed assays to determine if this was the case.

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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 with 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 pigment produced by the cells compared to uninduced Heme Oxygenase and the bacteriophytochrome.

The cells were lysed and the protein extract run on an SDS page gel. The gel showed the protein bands near the expected size (27 kDa). This, along with our pigmentation, showed that the BioBrick was producing the functional protein.

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Bacteriophytochromes Results

Like the Heme Oxygenase, the bacteriophytochromes from Deinococcus radiodurans and Agrobacterium tumefaciens were codon optimised for use in E. Coli. We did not use genomic DNA or previous Macquarie University team DNA at any stage, everything was performed with synthetic DNA we designed. The identity of the plasmid was determined by sequencing and by digestion. The bacteriophytochromes were characterised by demonstrating their ability to bind biliverdin and by their characteristic absorption profile.

The restriction enzymes used in this digestion were,

X= Xba1 P= Pst E= EcoR1 S=Spe1

We used a 1kb ladder (in increasing Kbp: 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10).

The digest above shows only shows one band for the different digests performed on the Agro T7 plasmid. The length of the vector and the bacteriophytochrome are similar and make it difficult to resolve the two bands. Only the digested plasmid can be seen which indicates that the bacteriophytochrome component was assembled correctly. Based on the length in base pairs of the fragments (2.25 kbp) we determined that assembly was successful. The first Agro with no T7 digest (asterixed band) shows incomplete digestion of a BioBrick plasmid. This enacted as a control and indicated the size of the plasmid.

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Bacteriophytochrome Sequencing

Plasmids extracted from the different transformations were sequenced.

Sequence	Expected Identity	Identity	E value	Max ID
F3CE1	Deinococcus radiodurans Bacteriophytochrome	Chain A, Crystal Structure Of A Monomeric Infrared Fluorescent Deinococcus Radiodurans Bacteriophytochrome Chromophore Binding Domain	3.00E-160	99%
F3CE2	Deinococcus radiodurans Bacteriophytochrome	Chain A, Crystal Structure Of A Monomeric Infrared Fluorescent Deinococcus Radiodurans Bacteriophytochrome Chromophore Binding Domain	2.00E-161	99%
F3CE3	Deinococcus radiodurans Bacteriophytochrome	Chain A, Crystal Structure Of A Monomeric Infrared Fluorescent Deinococcus Radiodurans Bacteriophytochrome Chromophore Binding Domain	8.00E-163	99%
R4KE2	Agrobacterium tumefaciens Bacteriophytochrome	bacteriophytochrome protein [Agrobacterium tumefaciens str. C58]	0	99%
R5CE1	Agrobacterium tumefaciens Bacteriophytochrome	bacteriophytochrome protein [Agrobacterium tumefaciens str. C58]	0	99%
R5CE3	Agrobacterium tumefaciens Bacteriophytochrome	bacteriophytochrome protein [Agrobacterium tumefaciens str. C58]	0	99%

We successfully produced the bacteriophytochromes with protein sequences matching the theoretical sequence. The difference in identity is due to the codon optimisation performed. It was also affected by short sequence reads. The 0 E value shows that we have produced the appropriate bacteriophytochrome.

The The Blastx pipeline showed that there was an identical match to the orginal source. This shows that the Gibson Assembly reactions were successful and we expect the protein to be functional. Blastn were run to determine the deviance from the theoretical sequence. The Blastn searches produced indicated that the sequences were nearly identical. The changed bases were examined on the sequencing output and determined to be possible misreads.

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Bacteriophytochrome Characterisation

We demonstrated that the bacteriophytochrome were functional by displaying their ability to bind biliverdin as well as by their presence in an SDS PAGE gel stained with Coomassie Blue.

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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 1 770 bp). 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.

The Gels

An SDS page gel was run of the ligation products to observe if the heme oxygenase was able to produce biliverdin and then to determine if it was binding with the bacteriophytochrome. The biliverdin binds to a specific site in the bacteriophytochrome. As biliverdin is fluorescent this coupling can be observed by irradiation with infrared (IR) light.

The SDS page gels for the Switch Constructs can be seen below,

The gel contains the following constructs:

The Protein Molecular Weight Marker 1C3C, the heme oxygenase and Deinococcus bacteriophytochrome ligation products (Lanes 2, 5, 6). 1C, the heme oxygenase (Lanes 3, 4) 4KE, Agro bacteriophytochrome (Lanes 7, 8).