Part:BBa_K4636040

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]

Main

The composite part, Insert_0101802, is composed of the following elements: a restriction site, a modified T7 promoter, the target sequence, and another restriction site. This composition is illustrated below. (Figure 1)

Figure 1. Schematic diagram of Insert_0101802.

In our project, we carried out a polymerase chain reaction (PCR) using the primers PCR_0101802fw (BBa_K4636003) and PCR_0101802rv (BBa_K463604) to amplify our target sequence. With these segments, we were able to perform two experiments. The first involved double digestion, while the second focused on in vitro transcription (IVT). After the double digestion, the insert could be ligated with the pUC19 vector and then transformed into E. coli for further cloning. In the case of IVT, the RNA product was used for circularization.

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 2. 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.

Experiment

1. insert digestion
Since the digestion site is included in our PCR primers, a successful digestion of the insert(Figure 3. lane 3) amplified by PCR_0101802fw and PCR_0101802rv would indicate the success of the primer design.

Figure 3. The agarose gel electrophoresis result of insert digestion. lane 1: 100 bp DNA ladder. lane 2: Insert_0004771 digestion product. lane 3: Insert_0101802 digestion product.

2. PCR
We used the PCR_0101802fw and PCR_0101802rv primers to amplify Insert_0101802. As demonstrated in the gel electrophoresis results (Figure 4), the band's correct position confirms the accuracy of our primer design.

Figure 4. PCR result of Insert_0101802. Lane 1: 50bps ladder, Lane 2,3: Insert_0101802

3. IVT
Since any remaining DNA templates had been degraded by treating DNase I, the presence of a band at lane 1 indicates a successful in vitro transcription (IVT) with the assistance of the Modified_T7 promoter. (Figure 5)

Figure 5. lane 1: low range RNA ladder, lane 2: Linear form RNA, lane 3: Circularization product, lane 4: Circularization product + RNase R, lane 5: Circularization product + RNase R

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