Results

We made these constructs to measure translation efficiency of our RBSs. We assembled LacZα to performed β-Galactosidase assay.

positive control

pTet-B0034-LacZα-dT

negative control

No insert

SD2

pTet-SD2-LacZα-dT (BBa_K1084121)

SD4

pTet-SD4-LacZα-dT (Bba_K1084122)

SD6

pTet-SD6-LacZα-dT (Bba_K1084123)

SD8

pTet-SD8-LacZα-dT (BBa_K1084124)

We moved our constructs to pSB3K3 plasmid, cultured for 9 hrs in 5 mL LB media round tubes, and performed β-Galactisidase assay. Reaction time was 2 hrs.

fig.1: Picture of β-Galactosidase assay. orange: strong LacZα expression, yellow: weak expression.

fig.2: LacZα expression. x axis: sample name, y axis: standardized expression (positive control = 1.0), bar: standard error. n=24.

LacZα protein hydrolyses substrate (Chlorophenol red-β-D-galactopyranoside), which is yellow. Product of this reaction has red color. Therefore, solution will turn deep orange if LacZα expression is strong (fig.1).

We measured absorbance of catalytic reaction solution at 595 nm, standardized using positive control and made into graph (fig.2). Construct with SD4 showed the strongest LacZα activity. Second strongest was the SD8, followed by SD6. SD2 had the weakest activity. There is a significant difference in translation efficiency of our RBSs. Translation efficiency of SD4 and SD8 is the same as BBa_B0034, which is most used RBS.