Parts-collection

EXTENDED NEURAMINIDASE VIRUS

During the initial period of our iGEM journey, we have successfully created 2 engineered NA proteins with elongated stalk domains : NAS14 (BBa_K4638006) and NAS28 (BBa_K4638007). These composite parts are made up of different basic parts that we found in both research papers and biobricks.

In our project, we hope to construct the neuraminidase-boosted influenza virus. We have adopted two methods to modify influenza virus, one of which is to insert amino acid segments from other influenza viruses into the stalk region of neuraminidase, so as to extend neuraminidase and make it more prominent on the surface of influenza virus.

The virus rescue system we used was the 12-plasmid system donated by Academician Gao Fu. We focus on the transformation of A6 plasmid in the 12-plasmids system. Insert of 14 amino acids from A/Tokyo/67 (H2N2) at position 42 as A6-SI14, and insertion of 28 amino acids from A/Tokyo/67 (H2N2) and A/Tern/Australia/G70C/75 (H11N9) at position 42 as A6-SI28.

1. NAS14 is made of :
		S14 (A/Tokyo/67 (H2N2) stalk) (BBa_K4638000) NAWT-A (BBa_K4638008) NAWT-B (BBa_K4638009)
2. NAS28 is made of:
		S14 (A/Tokyo/67 (H2N2) stalk) (BBa_K4638000) S28 (A/Tern/Australia/G70C/75(H11N9) stalk) (BBa_K4638001) NAWT-A (BBa_K4638008) NAWT-B (BBa_K4638009)

NAS14 is another name for SI14 (named in "Engineering" and “Wetlab”);NAS28 is another name for SI28 (named in "Engineering" and “Wetlab”).

MUTANT VIRUS WITH EXCHANGE PACKAGING SIGNAL

By exchanging the packaging signals of the neuraminidase and hemagglutinin RNA segments, we reversed the expression levels of neuraminidase and hemagglutinin, thereby achieving the objective of better displaying neuraminidase on the surface of the influenza virus. Additionally, we mutated the initiation codon ATG within the 5'-end packaging signal to TTG. Also, due to the presence of residual packaging signals in the coding regions of the HA protein and NA protein, we introduced synonymous mutations in this region to avoid the influence of the remaining packaging signal. Through homologous recombination, we successfully constructed the A6-HAps(mut) plasmid and A4-NAps(mut) plasmid.

1. EX-NA-MUT is made of :
		Mut-HA packaging sequence-5' (BBa_K4638014) Mut-NA WT (BBa_K4638016) HA packaging sequence-3' (BBa_K4638011)
2. EX-HA-MUT is made of:
		Mut-NA packaging sequence-5' (BBa_K4638015) Mut-HA WT (BBa_K4638018) NA packaging sequence-3' (BBa_K4638013)

EX-NA-MUT is another name for A4-NAps(mut) (named in "Engineering" and “Wetlab”); EX-HA-MUT is another name for A6-HAps(mut) (named in "Engineering" and “Wetlab”).