Part:BBa_K4829018:Design

IVT of this sequence produces mRNA coding for the CD36 membrane protein

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 527
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 165
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1349
1000
COMPATIBLE WITH RFC[1000]

Design Notes

We have optimised this sequence using the RiboTree software, developed by Das Labs. You may access the same on the dry lab page of iGEM23_IISc-Bengaluru 2023 and on the parts numbered BBa K4829019 and onwards.

This unique mRNA therapeutic design gives the bio-bricks registry a platform technology of mRNA-based therapeutics, something which was not there previously. The design of this part involved:

Using a slightly modified T7 promoter, keeping in mind the use of the correct capping reagents.
Optimising the sequence for human expression

Using a modified T7 promoter

The 'gold standard' of capping reagents of mRNA in the market at present could very well said to be the CleanCap®AG reagent. However, for use of this reagent, the last 2 bases of the T7 promoter must be AG and NOT GG. For this reason, we are also unsure if ARCA or other such capping reagents would work with the modified T7 promoter, as we have used only CleanCap®AG for our experiments.

Optimising the sequence for human expression

This was an easy task, done on TWIST biosciences codon optimisation software.

Tailing

There are 2 ways to incorporate the polyA tail of the mRNA: PolyA tailing kit of NEB after IVT of gene fragments/PCR product, or use a vector with the polyA tail already incorporated, linearise it and perform IVT. However, such vectors as mentioned are hard to come by. Ours was synthesised by Genscript and may be found on our Wiki.

Source