Team:CLSB-UK/Project/AmilCP

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AmilCP

Overview

BBa_K592025, the AmilCP blue-purple chromoprotein, was chosen for our project as a reporter protein. This was particularly important for our project because we wanted a strong colour, visible to the naked eye for simple confirmation of Synechocystis PCC sp.6803 transformation. The AmilCP protein has a maximum absorbance of 588nm^[1] making it easily visible without the use of ultraviolet light. It was also ideal for Synechocystis transformation because it has a very different colour to chlorophyll. We easily obtained BBa_K592025 from the registry and used it in our part BBa_K2078002 - K592025 ligated with the J23119 plasmid. We were able to use AmilCP to report that this part was expressed as expected and we have been able to use it to check that we are on track at regular intervals throughout the year.

Figure 1. AmilCP expression colour

Theory

AmilCP is a chromoprotein from the coral Acropora millepora which naturally exhibits strong color when expressed. The protein has an absorbance maximum at 588 nm giving it a blue/purple color visible to the naked eye, thereby requiring no instruments to observe. The strong color is readily observed in both LB or agar culture, in less than 24 hours of incubation. It is very similar to other coral chromoproteins in sequence; however, its absorption maximum (592 nm) is red-shifted by about 10 nm compared to many other coral chromoproteins, making the protein appear blue instead of purple to the naked human eye. The closest homolog of amilCP is gfasCP, in comparison to which the amilCP protein has only four amino acid substitutions: S64C, I162L, S183T and S229P (numeration according to GFP from A. victoria). The blue phenotype is due to the substitutions at two sites: S64C and S183T^[1].

Genetic Approach

K592025 was digested with EcoRI and PstI and then gel extraction was used to extract and purify the smaller AmiCP coding region. The ligation of this part with the promoter J23119 was carried out to create Part BBa_K2078002 in the pSB1C3 plasmid. Between the promoter and AmilCP part, a biobrick scar is formed (ctactagag) between the EcoRI and SpeI sticky ends. This plasmid carries the CmR gene for chloramphenicol acetyltransferase and thus carries chloramphenicol resistance. The construct was electroporated into E-Coli for amplification. Following Amplification, the construct was miniprepped and isolated from gel. The process was repeated three times to concentrate it for submission and insertion into the pDF plasmid.

Figure 1. AmilCP expression colour

Results

Our E.coli with the new part grew slower than other transformants and we were also unable to recover as much plasmid by miniprep. The use of the strong consensus promoter J23119 slows down expression times of the AmilCP part in E-Coli (normal colour expression is seen within 24hours)[1] whereas we only saw the very beginnings of colour expression after the same time. We observed no AmilCP expression in Synechocystis PCC sp. 6803 even after a week. The pDF plasmid we used has a promoter region that has been tested in Synechocystis in its backbone which means we were not able to use it to characterise our promoter in our model organism, however it also means we know that K2078002 was expressed. Therefore we can say with some certainty that AmilCP is not a suitable reporter protein for use in Synechocystis PCC sp. 6803.

References

• [1] http://parts.igem.org/Part:BBa_K592025
• [2] Diversity and Evolution of Coral Fluorescent Proteins Naila O. Alieva, Karen A. Konzen et al.