Result

-35 region randomization

Figure randomized promoter sequences.

We randomized -35 region by PCR primers with random hexamer region. The template DNA was consensus_promtoer-B0034-mRFP1-B0015 (about 1,000 bp). We assayed the constructed sequences and isolated 10 distinct promoters. We sequenced the randomized promoter sequences to confirm that only -35 regions was changed.

promoter assay; mRFP1, LacZ and Kanamycin resistance gene

mRFP1

Figure mRFP1 assay result

mRFP1 expressing JM109 colonies were resuspend to 2 ml LBC liquid culture. After cultivation (180 rpm shaking at 37C) for 12 hrs, we measured OD650 with micro titer plate reader. We avoided using 600 nm because mRFP1 absorbs 600 nm. mRFP1 expression was measured with FIM. All 10 of the promoters were characterized. Five promoters were used as a reference.

Reference promoters are following

• BBa_R0010: pLac
• BBa_R0040: TetR
• BBa_J23106: constitutive promoter family member (1185 arb. unit)
• BBa_J23112: constitutive promoter family member ( 1 arb. unit)
• Negative control: not protein expression construct

promoter selection by modeling

We chose 5 of 10 promoters by the value of theoretical transcription efficiency. This efficiency is affected by binding energy in our assumption.

Figure Theoretical transcription efficiency distribution

LacZα

We selected five promoters from our original family to model. LacZα Only these promoters were characterized using LacZ assay. LacZ (β-galactosidase) activity was measured with β-galactosidase assay kit. (OZ Biogenesis http://www.funakoshi.co.jp/data/datasheet/OZB/GC-10002.pdf ) DH5α strain was used.

These data was compared with modeling data (logarithm of transcription efficiency, t. e.). BBa_K1084010 and BBa_K1084009 couldn't be characterized by mRFP1 assay.

Figure β-galactosidase assay result

Figure Comparison of assay results and modeling data

Kanamycin resistance gene

Kanamycin resistance gene is expressed by these promoters as POK construct.