Part:BBa_K3889070

Double terminator for Bacillus subtilis Double terminator fusing B0010 aand nagP native terminator for use in Bacillus subtilis.

Secondary Structure

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Measurement

• [http://openwetware.org/wiki/Cconboy:Terminator_Characterization/Results How these parts were measured]

Improvement:

Introduction:

While engineering any new circuit, there is always a need for well-characterized and predictable parts. Not only should the circuit function as expected, but it should also be orthogonal to irrelevant cell processes, thereby increasing the need to have efficient production and, in some cases, more importantly, efficient termination. While there are several well-studied and efficient terminators for E.coli, we found no specific efficient single terminator on the iGEM registry that could stand out for B.subtilis chassis. Hence, we decided to improvise a terminator which might fulfil this gap.

Measuring efficiency:

The experiment is divided into two cassettes: one reference and the other is a test cassette containing a terminator whose efficiency needs to be determined as shown by Gale et al.[1].

Fig 1. Spacer Cassette for Terminator check

Fig 2. Spacer replaced by BBa_B0010

Fig 3. Spacer replaced by BBa_K3889070

The reference (Fig 1) and the test cassette (Fig 2 and 3) provide us with the expression levels of both the fluorescent proteins which could be compared to tell us how efficiently the terminator is working.

Formulae for terminator efficiency [1]:

where

mCherry₀ → mCherry produced by device without terminator

sfGFP₀ → sfGFP produced by device without terminator

Using the BBa_K3889130 without any changes, TE_Device can be calculated which gives the expression of mCherry in absence of terminator.

where

mCherry → mCherry produced by the device with terminator that needs to checked

sfGFP → sfGFP produced by the device with terminator that needs to checked

d-score

For E. coli terminators d'Aubenton Carafa [3] gave a scoring system as shown below:

where

d is the d-score

Δ G is the Gibbs free energy of stem-loop formation in kcal/mole

n_SL is the length of the stem-loop

T-Score is the score for the T-stretch of the terminators

Coefficients are according to fitting the d'Aubenton Carafa’s model

The T-Score is calculated as follows:

Where

x₀ = 0.9

x_i = 0.9 if i^th nucleotide is thymine

x_i= 0.6 * x^i-1 if i^th nucleotide is not thymine