Team:HK HCY LFC/Parts
Overview for Our Parts
Figure 1: A Drawing Showing the Biobricks in the Plasmid. BBa_K2618000, BBa_K2618001 and BBa_K2618002 are the insert double strand DNAs that responsible for synthesis of the 3 strands in our nanomachine respectively. Each plasmid will hold one insert. One insert contains sequence of promoter, single strand DNA, HTBS and terminator.
1. Promoter
Promoters contain specific DNA sequences that provide an initial binding site for RNA polymerase and for proteins called transcription factors that recruit RNA polymerase.
2. Terminator
Terminator is a section of DNA sequence that marks the end of a gene in genomic DNA during transcription, by providing signals in the newly synthesized transcript.
3. HTBS
The transcribed RNA will have an HTBS sequence contained in it. HTBS, stands for HIV transcriptase binding site, recruits HIV reverse transcriptase (HIVRT), which is a kind of reverse transcriptase. Reverse transcriptase performs the conversion of RNA into DNA naturally in retroviruses, which possess RNA genomes that need to be altered to DNA before integrating into a host genome. [1] Therefore, HIVRT can reverse transcribe the ncRNA produced into a single strand DNA.HIV reverse transcriptase is used in the project not only because of it being a subject of intensive research [1], but also its higher thermostability. M-MLV reverse transcriptase and AMV RT are another reverse transcriptase that are applied in laboratory. Compare with the two RTs, HIV RT performs as well as M-MLV RT and AMV RT at 37°C and 42°C. However, the enzyme retains a significantly higher fraction of its activity at 50°C than the other RTs. [2]
Figure 2: Overview of the cloning process
Figure 3(a): Flowchart Showing the Expression of Plasmid
Figure 3(b): Expression of plasimid by E.coli
In the insert DNA, one strand is recognized as template strand while the another is recognized as coding strand. Coding strand, running from 5’ to 3’, corresponds to the same sequence as the RNA, and the same sequence as the ssDNA aimed to synthesis. Template strand, running from 3’to 5’, is the strand where RNA polymerase will be attached during transcription. If the recombinant plasmid have been successfully taken up by the E. coli, RNA polymerase first bound to the initiation site of template strand and began transcribing in the 5′ to 3′ direction [2]. The DNA sequence between promoter and terminator of template strand would be transcribed into RNA by RNA polymerase in E. coli. The transcribed RNA contains two parts: the sequence which is complementary to the single strand DNA and the HTBS sequence. The formation of single strand DNA require the E. coli to possess a HIV transcriptase, which can bind to the HTBS site of the transcribed RNA. Reverse transcription is then performed by the HIV transcriptase to reverse transcribe the RNA, in a 5' to 3’ direction [1]. Finally, the strands are cleaved by RNase to produce the single strand DNA.
References
[1] Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7, 11179.
[2] ThermoFisher Scietific. Recombinant HIV Reverse Transcriptase. Retrieved Oct 2, 2018, from https://www.thermofisher.com/hk/en/home/references/ambion-tech-support/probe-labeling-systems/tech-notes/recombinant-hiv-reverse-transcriptase.html
Please include a table of all the parts your team has made during your project on this page. Remember part characterization and measurement data must go on your team part pages on the Registry.
| Type | Name (Part number) | Type | Description | Length(bp) |
|---|---|---|---|---|
| Basic Part | BBa_K2618000 | DNA | ssDNA for DNA tweezers assembly (Strand 1) | 386 |
| Basic Part | BBa_K2618001 | DNA | ssDNA for DNA tweezers assembly (Strand 2) | 359 |
| Basic Part | BBa_K2618002 | DNA | ssDNA for DNA tweezers assembly (Strand 3) | 362 |
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