DNA

Part:BBa_K4636040:Design

Designed by: Lo-Chueh, Chu   Group: iGEM23_NTHU-Taiwan   (2023-10-11)


Insert_0101802


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

For two restriction sites design, we should consider following tips[1], [2], [3]:

(1) Sitting sequence (4~5 b.p.): Located Outside of the restriction site and responsible for enzyme activity. We should avoid the same sequence as restriction sites, and it must not contain GG or CC sequence.
(2) Restriction sites can’t be found in insert.
(3) Use two different restriction sites with distinct restriction enzymes.
(4) The two different restriction enzymes should have similar reaction temperatures and use the same reaction buffer.
(5) Both of the restriction enzyme have high activity in the same reaction buffer.

Refer to previous study[4], [5], we add four parts of sequence to enhance the transcription ability:


Figure 1. Modified T7 promoter.

(1) Upstream of the T7 promoter, from -18 to -21, there is a high AT-rich region. This enhances the binding affinity of the T7 polymerase for the DNA template.
(2) Upstream of the T7 promoter, from -22 to -27, there is a highly GC-rich region. This enhances the binding affinity of the T7 polymerase for the DNA template.
(3) Downstream of T7 promoter from +1 to +3 : With triple G (GGG) sequence. It can increase binding affinity of the T7 polymerase for the DNA template. (Transcription start from the first G)
(4) Downstream of T7 promoter from +4 to +8 : With highly AT rich. It can assist DNA template to form a initiation bubble which increases the initially transcription ability.

Reference

1. https://www.addgene.org/protocols/primer-design/
2. https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
3.https://www.researchgate.net/post/Are_the_sitting_sequences_added_before_the_PCR_primers_arbitrary_or_specific_to_restriction_endonucleases
4. Tang, G. Q., Bandwar, R. P., & Patel, S. S. (2005). Extended upstream A-T sequence increases T7 promoter strength. The Journal of biological chemistry, 280(49), 40707–40713. https://doi.org/10.1074/jbc.M508013200
5. Conrad, T., Plumbom, I., Alcobendas, M., Vidal, R., & Sauer, S. (2020). Maximizing transcription of nucleic acids with efficient T7 promoters. Communications biology, 3(1), 439. https://doi.org/10.1038/s42003-020-01167-x