Result

-35 region randomization

We randomized -35 region by PCR primers with random hexamer region. The template DNA was consensus_promtoer-B0034-mRFP1(E1010)-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. Our consensus promoter is K1084001.

promoter assay; mRFP1, LacZ and Kanamycin resistance gene

mRFP1

mRFP1(BBa_E1010) 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 fluorescence imaging machine. All 10 of the promoters were characterized. Five promoters were used as a reference. BBa_K1084010 and BBa_K1084009 couldn't be characterized by mRFP1 assay because of mutation at CDS.

Reference promoters are following

• BBa_R0010: pLac
• BBa_R0040: pTetR
• 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 (for theoretical explanation, see "Method page"). This efficiency is affected by binding energy in our assumption.

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.

Reference promoters are following

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

Kanamycin resistance gene

Kanamycin resistance gene is expressed by these promoters as Promoter Selector construct.

As shown in the Shuffling_Kit/Examples page, Kanamycin resistance also differed by promoter. In the result, K1084010 has the best Kanamycin resistance activity. However, the result is containing some controversial problems, such like differences of concentration among used DNA solutions at ligation step, or the Kanamycin resistance measuring problem: the Kanamycin resistance gene activity wouldn't be measured by just counting colonies on a plate. There are needed more intermolecular assay for measuring the enzyme activity.

Comparison of assay results (Conclusion)

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 because of mutation at CDS.