Part:BBa_K2054003:Experience

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Applications of BBa_K2054003

Plasmid Construction

We first digested the G-block fragments we ordered and the linearised pSB1C3 backbone by PstI and EcoRI. Then we put them together via ligation at a molar ratio (backbone:insert) of 1:1, 1:3 and 1:10. Below a 1% Agarose Gel to show the ligation (1:3 ratio).



(lanes concerned: lanes 4, 7 and 11)

The ligated products (backbone-to-insert ratios of 1:10 and 1:3 and 1:1) were then transformed following the protocol available on NEB. The ligated pSB1C3 digested with PstI and EcoRI was transformed as an negative control, where no colonies were observed on the corresponding plate. There were also no colonies on the plate for ratio of 1:1.

 On the other hand, considerable numbers of colonies were observed on the plates with the plasmids shown above (1:3). It was mini-preped on the following day after another 14-16 hrs of incubation in LB broth. We performed a colony PCR (and inoculate the colonies in the meantime) to select the appropriate ones using the NEB protocol. A 1% agarose gel electrophoresis was also performed right after to determine which colonies contain the right inserts-ligated plasmids. We picked 4 colonies per plate. The couple of primers used was insert-specific, which will amplify the inserts, having expected band sizes of 2375bp (plasmid) and 398bp (insert).

1% Agarose of the PCR products, with the colonies labelled. (lanes concerned: lanes 11-15)

This was from ligation with backbone-to-insert ratios of 1:3.

 Colonies 1a-1d, 2b-2d, 3a-3d, 4b-4d, 5a-5d appeared to be the appropriate ones. We selected those with weaker intensities from these for inoculation into the LB broth (with 20ug/mL Chloramphenicol added) and incubated them overnight at 37°C. Mini-prep was performed meanwhile followed by a 1% gel electrophoresis.


(lanes concerned: lanes 6-7)

Multiple bands were seen in each lane, and this could be due to different configurations of the plasmids which affect the migration through the gel pores. E.g. in the 2nd lane, the plasmid p_O1 might exist in super-coiled, linearised and circular (nicked circle) configurations which gave rise to the lower, the middle and the upper bands. The middle band matches the band sizes of the ladder on the left, which is around 2300-2500bp.

Diagram to show the migration of different configurations of plasmid of our plasmid p_O1

To double check whether the above was the case and whether the mini-preped plasmids are the real deals, an RE digestion with EcoRI was performed following the protocol from NEB followed by an 1% Agarose Gel Electrophoresis.

(lane concerned: lane 4)

PCR was also performed following the protocol available in NEB with insert-specific primers such that inserts (approximate sizes) would be amplified. Below shows the 1% Agarose Gel electrophoresis of the PCR product. It is expected to have band sizes mentioned above.

1% agarose gel stained with GelRed and visualised under UV light to show PCR products of mini-preped plasmids. (lanes concerned: lanes 6-7)

The following schematic diagram (fig 1a extracted from the article) summarizes the idea of our project.

source: Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7.

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