Aim: To characterise the T1 terminator from E. coli rrnB, K1725081, for submission to the registry. Results Overview: K1725081 was successful in terminating transcription to the same level as B0010 and B0015. Basic Parts submitted: • BBa_K1725081
In prokaryotes, such as E. coli, terminators occur at the end of a coding region or operon, and stop transcription. K1725081 was used in our Bistable Switch.
To characterise K1725081, the plasmid backbone pSB1A10 was used (figure 1). pSB1A10 contains I0500 (pBAD/araC), B0034 (strong ribosome binding site), E0040 (GFP), BioBrick prefix, BioBrick suffix, B0034, E1010 (RFP). With no terminator between the BioBrick prefix and suffix, cells transformed with the plasmid would fluoresce both green and red, however, when a terminator is inserted between the prefix and suffix, RFP expression is reduced, and the cells would fluoresce green.
Plasmids for testing terminators were assembled by BioBrick Assembly: K1725081 in pSB1A10, B0015 (a double terminator consisting of B0010 and B0012; the most commonly used terminator in the registry, it is considered to be reliable) in pSB1A10, B0010 (the terminator K1725081 is based on) in pSB1A10, and pSB1A10 with no terminator - the latter was made by ligating the XbaI and SpeI sites in the plasmid backbone prefix and suffix together (so the BioBrick cloning site became EcoRI-scar -PstI).
The results of the fluorescence scan are shown in Figure 2. K1725081, B0015, and B0010 all show reduced RFP expression, indicating that they are successfully stopping transcription after GFP - although neither of these terminator stop 100% of transcription, so there is still some RFP expression for all three.