Part:BBa_K4829016:Design

IVT of this sequence produces mRNA coding for an scFv antibody blocking PD1

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 558
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 273

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:

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

Selecting the signal peptide

This protein is NOT HUMAN. As a result, it does not have a standard signal peptide for secretion, and a chimeric protein must be made if this protein is to be secreted out of the cell. The list of most commonly used signal peptides as on (https://novoprolabs.com/support/articles/commonly-used-leader-peptide-sequences-for-efficient-secretion-of-a-recombinant-protein-expressed-in-mammalian-cells-201804211337.html) is a rather long one, and to select the best one for our protein(the antibody), we used the software SignalPeptide6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/). The signal peptide of Albumin(HSA) was selected after appending the various signal sequences listed on the page to the beginning of the antibody sequence.

 Figure 1. Probability of cleavage and secretion via Sec/SP1 secretion pathway with the Albumin(HSA) SP: Cleavage site between pos. 16 and 17. Probability 0.980935

This was in comparison to the probability of cleavage with CD33 and tPA

 Figure 2. Probability of cleavage and secretion via Sec/SP1 secretion pathway with the CD33 SP: Cleavage site between pos. 16   and 17. Probability 0.967106

 Figure 3. Probability of cleavage and secretion via Sec/SP1 secretion pathway with the tPA SP: Cleavage site between pos. 22 and 23. Probability 0.965749

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

This is a composite biobrick of both human(CD33 SP) and non-human sequences.