Part:BBa_K3889070

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

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

This scoring system was modified by de Hoon et al. [2] for Bacillus subtilis as per their model which is as follows:

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 de Hoon model [2]

The T-Score is calculated as follows:

where

λ ₀ = 0.144

δ _i = 0 if i^th nucleotide is not thymine

δ _i = 1 if i^th nucleotide is thymine

As the d-score takes into account the Gibbs free energy, length of the stem-loop and the richness of thymine in T-stretch which are essential for a rho independent terminator. Hence, the d-score can provide a rough idea about how good a terminator is. In other words, the higher the d-score higher will be the terminator efficiency.[3]

Improvement

We decided to improve BBa_B0010 in order to make a strong terminator which can be used for primarily the B.subtilis chassis while still retaining its efficiency in E.coli. For doing this we modified the tail of Bba_B0010 and fused another Rho-independent terminator from the Bacillus subtilis genome on the basis of its d-Score.

From a list of 425 native B.subtilis terminators taken from the study conducted by Michiel et al [2], we calculated the d-score of each terminator to get a rough idea of their efficiency. Based on the results the highest d-score= 5.666126119 was of the terminator belonging to the gene nagP. Both BBa_B0010 and nagP terminators were ligated to form a double terminator.

Based on our calculations, we decided to go with nagP terminator. We modified the end regions of BBa_B0010 and ligated to it the nagP terminator to create an improved version

Using the server RNAFold [4] we calculated the minimum energy to show in silico that the improved terminator will have more negative Minimum Free energy as shown:

	BBa_B0010	BBa_K3889070
Minimum Free Energy (kcal/mol)	-40	-64.6

The predicted structure for these two terminators as given by RnaFold server is: BBa_B0010:

BBa_K3889070: