Part:BBa_K2100068
pENTR L7Ae
This entry vector contains L7Ae, which is used in conjunction with k-turns to suppress expression of genes. Unlike DNA-level transcriptional repressors, L7Ae acts as a post-transcriptional repressor by binding instead to mRNA. After transcription, the k-turn sequences form small loops in the mRNA that L7Ae can recognize and tightly bind to. When L7Ae is bound to mRNA, the mRNA cannot bind to a ribosome, thus inhibiting translation of the mRNA. this part has been improved by iGEM AFCM-Egypt 2021 team by fusing it with dCas13 via Gly Ser linker.
Improvement & Characterization by AFCM-Egypt 2021
Improvements To Part by addition of cas12g
AFCM-Egypt 2021improved this part by utilizing The Gly Ser linker to conjugate it with the L7Ae protein (which inhibits transcription by binding to its kink-turn). The mechanism simply identifies and attaches to mRNA in the cancerous environment, causing the L7Ae protein to be consumed. As a result of not binding to kink-turn, the inhibitory action on transcription is inhibited, resulting in transcription activation and a rise in vaccine yield. As a result, it is a cell-specific design that binds to PD-L1 mRNA, which plays an immune evasion role in cancerous environments, particularly TLCs.As shown in part BBa_K3743015
Improvements To Part by addition of dcas13
AFCM-Egypt 2021improved this part by a deactivated Cas13 used in conjugation with L7Ae protein (that has an inhibitory effect on the transcription by binding to its kink-turn) by Gly Ser linker. The system simply recognizes and binds to mRNA in the cancerous environment which leads to consumption of L7Ae protein. Therefore, not binding to kink-turn to inhibit the inhibitory effect on the transcription which finally leads to stimulation of the transcription in order to increase the yield of the vaccine. That is why, it is a cell specific design by binding to mRNA of PD-L1 which has an immune evasion role in the cancerous environment especially TLCs.As shown in part BBa_K3743007
Characterization Of Mutational Landscape
After performing mutagenesis prediction of mutational landscape of L7Ae and tested the effect of these mutations on the evolutionary fitness of the protein after generating multiple sequence alignment of the protein sequence and predict mutational landscapes. As shown in the chart, the (G108S) mutation showed the highest score compared to other mutations. On the contrary, we can see that the (G108K) contributed to the lowest evolutionary fitness to L7Ae. As shown in Figure (1)
Characterization by Mathematical Modelling
In order to simulate the dynamics of the used riboswitches which acts as an essential safety switches in our vaccine design, Mathematical modeling is performed using ordinary differential equations (ODEs) and fitted parameters.
As shown in Figure(2), P2 which represents the binding state shown inhibition throughout the time which conclude inhibition of the circuit. On the other hand, P1 which represents removing the inhibitory effect of riboswitches showed increased transcription of the circuit which reach the steady state after about 200 seconds.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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