Artificial transcriptional terminators

The goal is to create a series of transcriptional terminators with varying efficiencies. The majority of transcriptional terminators have a G+C rich stem of 7(+/-1)bp and a loop of 4(+/-1) nucleodtides followed by a poly(U) tail. Two common loops are UUCG and GAAA, both of which are known to increase RNA hairpin stability. The sequence GCGGG(G) is a common sequence found on the 3' arm of the stem. [1]

Effects of stem loop sequence on terminator efficiency

Bulges and mismatches in the stem, as well low G+C content of the stem will lower TE more than reducing the length of or elimination of the poly(U) tail [2]. The sequences downstream of the poly(U) tail and between the stop codon and the start of the stem loop structure also affect the TE of a terminator, particularly T7Te or T3Te.

• T7Te

Several sources [3] [Chamberlin 79] measured the termination efficiency(TE) of T7Te at around 90%. However, efficiency for the biobricks part BBa_B0012 [1], also T7Te, is around 30%. T7Te has a very short poly(U) tail and requires the further downstream sequence for efficiecent termination [3], and this further downstream sequence is lacking in BBa_B0012. If the sequence for BBa_B0012 is lengthened to include this downstream segment, then the TE of part should be improved.

Predicting terminator efficiency

It may be possible to predict terminator efficiency using methods from d'Aubenton, in particular, the score d assigned to a possible terminator sequence

d = nt*18.16+Y*96.59-116.87

where nt measures the statistical distribution of the T residues in the non transcribed DNA strand and Y is the free energy per nucleodtide of the stem loop structure.

The score d will give a rough estimate of how efficient a terminator is.

d<0: TE<20%

0<d<30: 20%<TE<70%

d>30: TE>70%

Ideal terminator

• has 6 base stem with 3' sequence of GCGGGG
• 4 base loop, either UUCG or GAAA
• tail containing >8 uridines
• for a biobrick part, flanking regions will be biobrick site

Designed terminators

The score d is calculated as mentioned above. The energy of hairpin formation, delG, is caluclated using UNAFold.

These terminators are designed to be bidirectional. delG reverse is the energy of hairpin formation on the opposite strand.

• Terminator 1:

delG=-12.6 d=59.31 %T>90

delG reverse=-10.2 d=44.82 %T>90

stem loop: CCCCGCTTCGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCAAAAAAAAACCCCGCTTCGGC

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTAGCGGCGAAAAAAAAACCCCGCCGAAGC

• Terminator 2:

delG=-12.6 d=35.78 %T>90

delG reverse=-10.2 d=21.28 %T=75

stem loop: CCCCGCTTCGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCCGCAAAAAACCCCGCTTCGGC

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTAGCGGCGGCGAAAAAACCCCGCCGAAGC

• Terminator 3:

delG=-12.6 d=26.12 %T=80

delG reverse=-10.2 d=11.64 %T=40

stem loop: CCCCGCTTCGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCCGCCAAAAACCCCGCTTCGGC

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTAGCGGCGGCGGAAAAACCCCGCCGAAGC

• Terminator 4:

delG=-12.6 d=15.40 %T=55

delG reverse=-10.2 d=0.91 %T=<20

stem loop: CCCCGCTTCGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCCGCCGAAAACCCCGCTTCGGC

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTAGCGGCGGCGGCAAAACCCCGCCGAAGC

• Terminator 5:

delG=-12.6 d=3.49 %T=25

delG reverse=-10.2 d=-11 %T<10

stem loop: CCCCGCTTCGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCCGCCGCAAACCCCGCTTCGGC

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTAGCGGCGGCGGCGAAACCCCGCCGAAGC

• Terminator 6:

delG=-16.2 d=54.38 %T>90

delG reverse=-18.9 d=66.22 %T>90

stem loop: CCCCGCCCCUGACAGGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAG

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTA

• Terminator 7:

delG=-16.2 d=30.84 %T=80

delG reverse=-18.9 d=42.69 %T>90

stem loop: CCCCGCCCCUGACAGGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCCGCAAAAAACCCCGCCCCUGACAGG

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTA

• Terminator 8:

delG=-16.2 d=21.19 %T=70

delG reverse=-18.9 d=33.03 %T=80

stem loop: CCCCGCCCCUGACAGGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCCGCCAAAAACCCCGCCCCUGACAGG

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTA

• Terminator 9:

delG=-16.2 d=10.46 %T=40

delG reverse=-18.9 d=22.31 %T=75

stem loop: CCCCGCCCCUGACAGGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCCGCCGAAAACCCCGCCCCUGACAGG

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTA

• Terminator 10:

delG=-16.2 d=-1.45 %T<10

delG reverse=-18.9 d=10.40 %T=40

stem loop: CCCCGCCCCUGACAGGGCGGGG

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCGCCGCCGCCGCAAACCCCGCCCCUGACAGG

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTA

• Terminator 11: this is not supposed to work. if it does, then something is wrong

delG=-3.3 d=-52.65 %T=0

delG reverse=-0.5 d=-69.56 %T=0

stem loop: TTTTATGAAAATAAAA

primer 1: GTTTCTTCGAATTCGCGGCCGCTTCTAGAG

primer 2: GTTTCTTCCTGCAGCGGCCGCTACTAGTA

References

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