Part:BBa_K1777016:Design

Device to produce circRNA

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 462
  Illegal XhoI site found at 833
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 804
  

Design Notes

This device is specially designed based on back-splicing mechanism. Here is the part which can form the circRNA to 'absorb' the miR-21 as a sponge. Therefore, the inverted repeat sequences should be added into unstream of 5 prime and downstream of 3 primer of the circlization sequence. We inserted the reverse-complementary repeat sequence via enzyme-digestion and T4-liagtion, while the direct repeat sequence cannot be inserted in the same way. We use Gibson Assembly instead.

Gibson Assembly

Because of the limited time, we cannot get the kit from NEB® and have to prepare the reagents by ourselves.

Compositions of Master Mix

Prepare 6 ml of 5X ISO Buffer in a 15 ml falcon tube as follows:

3 ml	1 M Tris-HCl pH 7.5
150 µl	2 M MgCl2
240 µl	100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)
300 µl	1 M DTT
1.5 g	PEG-8000
300 µl	100 mM NAD

add ddH₂O to 6 ml, store at -20°C in 320 µl aliquots.
Prepare 1.2 ml of Gibson assembly master mix as follows:

320 µl	5X ISO Buffer
0.64 µl	10 U/µl T5 exonuclease
20 µl	2 U/µl Phusion polymerase
160 µl	40 U/µl Taq ligase

add ddH₂O to 1.2 ml, store at -20°C in 15 µl aliquots.
The reagents are based on Miller Lab's compositions[2].

Protocol of Gibson Assembly

• Design the primers typically 40 bp in length (20 bp of vector and 20 bp of insert) and order them.
  
• PCR your gene of interest by the primers below.
  
• When PCR is running, linearize the vector with the enzyme digestion, followed by PCR clean up or gel extraction.
  You can save the linearized vector at -20°C for a few months before use. You can prepare the linearized vector by PCR if an appropriate restriction site is lacking.
• Prepare the insert and vector mixture with 1:1 insert vector molar ratio approprately.
  
• Mix 3.75 µl of Gibson master mix below with 1.25 µl of DNA mix in a PCR tube, and incubate samples in a thermocycler or a heater at 50°C for 60 minutes. After incubation, store samples on ice or at -20°C for subsequent transformation.
• Transform 2 µl Gibson reaction into 50 µl 5-α Competent E. coli cells.

Tips

• With a little excessive insert fragments with 1.05:1 insert vector molar ratio, the transformation efficiency tended to be better.

Source

We build this two device with BBa_K1777015 and BBa_K1777003, as well as beta-globin intron(BBa_K404107). We used the β-globin intron to increase the expression level of mRNA and provide SA and SD site to help the cirlization of mRNA at the same time.

References

[1] D Gibson.One-step enzymatic assembly of DNA molecules up to several hundred kilobases in size.Protocol Exchange.2009.
[2] [http://miller-lab.net/MillerLab/protocols/molecular-biology-and-cloning/gibson-assembly/ Miller Lab's Gibson assembly protocol]