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:
5X ISO Buffer
1 M Tris-HCl pH 7.5
3 ml
2 M MgCl₂
150 µl
100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)
240 µl
1 M DTT
300 µl
PEG-8000
1.5 g
100 mM NAD
300 µl
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].
Reference
[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]
Pick a restriction site (or a pair of restriction sites) where you want to insert the gene of interest (GOI) in. Design the primers using the convenient NEBuilder tool. Make sure to check the reading frame! Alternatively, you can design the primers by yourself. I usually construct the sequence map of the desired construct first and simply copy and paste from the sequence map. 20nt overlap with the vector region is more than enough; 15nt should work as well.
Order the primers.
PCR your GOI using the designed primers.
At the same time, linearize the vector with the restriction enzyme, 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.
Mix the insert and vector at appropriate molar ratios. 3:1 insert vector ratio is recommended if you are assembling two fragments. For more than one inserts, I usually keep all the inserts 3-fold more excess than the vector. If you don’t have time to quantify your DNAs, simply mix 1 µl of each fragment, works most of the time in my hands.
Mix 3.75 µl of Gibson master mix with 1.25 µl of DNA mix in a PCR tube, and incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at -20°C for subsequent transformation.
Transform 2 µl Gibson reaction into 50 µl 5-alpha Competent E. coli cells. Homemade Top10 works as well.
Recover the cells in SOC medium for 1 hr before plating.
16 hrs later, screen colonies by colony PCR, usually two colonies are sufficient for two way assembly.
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.
