Team:Uppsala/Improve

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In 2011 the iGEM Uppsala team managed to create an amilCP biobrick (BBa_K592009), a great addition to the iGEM repository of chromoproteins. When the part, which originates from a coral named Acropora Millepora, gets expressed it produces a strong natural blue colour that is very much visible to the naked eye [1].

In 2017, the Austin University, Texas iGEM Team decided to improve the part hoping for stronger expression resulting from codon optimization. By performing codon optimization they hypothesised that the seemingly unstable amilCP from 2011 would become more stable resulting in less white cultures on the plate, hence higher protein yield. The output part after optimization can be found in the registry as BBa_K2253002 [2].

Figure 1. The image above shows various streaked out colonies from the original blue chromoprotein, BBa_K592009. Both phenotypically blue and clear colonies grew, supporting the hypothesis that the blue chromoprotein is unstable and breaks or changes color at different rates. Plate streaked by Microbe Hackers 2016 Chromoprotein Team [2].

When studying the sequence, what's worth noting is that the sequence provided contains a frameshift that would suspectedly lead to a faulty protein that most likely won’t be expressing any colour, something that could be consistent with the shown result in figure 2. Figure 2 shows one single successful transformated colony which in that case might have been caused by a deletion mutation. In addition we can never know for sure if Austin Texas 2017 iGEM Teams optimization actually is better than the original part since a direct comparison in picture is missing on both the wiki and the part registry.

Figure 2. The image above shows the transformation plate of the electroporation of the codon-optimized blue chromoprotein. The blue colony shown was used to make overnight cultures to miniprep and sequence [1].

Original AmilCP sequence (BBa_K592009) was optimized for expression in E.coli K12 strain using the IDT tool for codon optimization. The sequence were then put in front of a strong constitutive promoter and RBS along with a double terminator. The sequence, and the original sequence with the same elements was then ordered as customized gene fragments from IDT along with another optimised sequence where the COOL software was used and thereafter it was optimised manually.

Unfortunately IDT wasn’t able to synthesise the original Austin Texas sequence even though multiple attempts were made. All of the times a base was switched resulting in an early stop codon. Hence we never was able to compare our optimized sequence with the sequence form the Austin Texas optimization. We continued with our experiment and decided to make a stability assay where we compare the expression between our two optimized AmilCP sequence and the original part from igem registry.

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Here is a second paragraph with E.coli written in italics. Any found genes which display promise will have to be validated by qPCR (which is a similar method) in a second run to confirm that they are only expressed due to the strongyle presence. Another approach to tackle our challenge is to screen for interaction between the surface proteins on the strongyle and short peptides. Through affinity screening of a random peptide library displayed on the surface of phages, we can select a peptide with a high affinity to the nematodes surface.