Part:BBa_K1223014:Experience

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Applications of BBa_K1223013 and BBa_K1223014

This part was used by us to incorporate the unnatural amino acid propargyl-L-lysine into various proteins.

For the characterization of the generic code expansion machinery parts we designed and carried out several experiments:

1. To determine and characterize our ability to incorporate an unnatural amino acid (Progargyl lysine) into a model protein site specifically we first used standard mutagenesis methods to add TAG stop codons to various sites in our model gene. Next we transformed the bacterium with our model protein on an expression plasmid and then transformed the same bacteria with an expression plasmid containing our PylRS and tRNAcua. We have grown bacteria in LB broth overnight with 1mM of expression inducers (IPTG) and 1mM of Propargyl lysine (UAA). Upon incubation we lysed the bacteria and performed a 'click' reaction with a fluorescently labeled azide (protocols are available on our team's wiki) to determine the presence of propargyl lysine in place. In order to visualize the protein, we ran the lysates in an SDS fluorescent PAGE.[7]

H117,N262,D411,M412 - position and amino acid that was replaced with the UAA. CueO - native protein without UAA incorporated.

In this fluorescent SDS PAGE image, it is shown that we were able to incorporate our UAA with high yields into different sites of the protein. H117, D411, M412 are different mutants were the natural amino acid was replace with an UAA.

Genetic code expansion machinery characterization

To determine selectivity and fidelity of our part we designed experiments were we aim to achieve selective progaragyl-l-lysine (UAA) incorporation into a model protein (CueO-6xHis). By selective we mean that when there is no proparagyl-l-lysine (Like in nature) only the truncated form of the protein will be expressed.

1st experiment:

Fluorescent western blot image, using anti-6xHis antibody to visulaize different CueO mutants expressed in the presence (+) and in the absence (-) of UAA. Commassie staining

Commasie stained gel image of the same samples shown on the western blot in figure 5.

In this experiment we produced both fluorescent western blot and coomassie stained gel (of the same experiment) that shows the specificity of the unnatural amino acid incorporation machinery in four different mutants in the E.coli copper oxidase (CueO). For each mutant we induced the production of the protein (~57kDa) with (+) or without (-) the unnatural amino acid present in the growth medium.

It can be seen very clearly from the fluorescent blot that the protein is present only in the culture that were incubated with the unnatural amino acid - except for G450 which appears to be a non-permisive site where there is no incorporation of UUA at all.

2nd experiment:

In this Anti-his-tag western blotting we were able to show that there in minimal non- specific incorporation when there were no UAA. In other words, our logic AND gate 2nd condition is robust. When our bacteria are in nature they could not translate their essential protein, but its inactive truncated form.

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