Difference between revisions of "Team:Fudan-TSI/Design"

The initiation of reverse transcription requires its cognate tRNA primer. For MMLV-RT, the primer is tRNA^Pro(AGG)⁶. After comparing its sequence to the endogenous tRNA sequences in E. coli, we found very little homology between them, which again proves our system’s orthogonality to prokaryotic system. However, this also arises problems regarding its post-transcriptional processing. To make sure that the exogenous tRNA could be successfully expressed and processed in E. coli cells into its matured form, we placed it under two promoters, one T7 promoter, and another pGlnW promoter, which is the endogenous promoter for GlnW and its downstream MetU. The CCA motif is added to the 3’ end, and followed by the interval sequence between two tRNA^Gln, and a T7Te terminator.

Apart from using the native tRNA^Pro, which requires an additional PBS outside the target sequence, we have designed a software tool which can design novel tRNAs aligning with the target sequence.

EGFP is cloned in the place of the reverse transcriptase, and fluorescence level is measured.

The gag-pol polyprotein is induced and SDS-PAGE is run to see if it has been successfully processed by the protease.

When Cre is not present in the cell, the system’s outcome would be reverse transcribed double-stranded cDNA. After lysing the cell and extracting its DNA components within, PCR amplification of the cDNA product would be performed and after electrophoresis, we could observe a brighter band if RT is induced to express compared to RT non-existent cells. For more quantitative analysis, qPCR would be performed.

Cre recombinase is derived from P1 bacteriophage, which could initiate recombination events between two loxP sites. Cre recombinase binds to the palindromic sequence in loxP, and after forming a tetramer (two Cre on one loxP), its active site would initiate different recombination process based on the orientation of the two loxP¹². Two loxP sites in the same direction would initiate self-splicing, resulting the sequence in between be excised and form a circular loop. If the two loxP sites are oriented opposingly, the sequence in between would be inverted. If the two loxP sites are placed on different sequences, the two sequence would be transferred into each other’s place, but at a lower efficiency than the other two events. Our system utilizes its strand-transfer recombination activity to insert the mutated target into its original place, thus replacing the unmutated version and allows for another round of mutation.

The expression of Cre recombinase is placed under a different operon, and anhydrotetracycline (aTc) serves as its inducer. The reason behind placing RT and Cre under different control has been elaborated in our modelling. Apart from the need of different final concentration of these two proteins for the system to achieve its optimal function, putting them under different promoters also enables better control over the system’s status.

Cre recognizes loxP sites and after forming a tetramer, initiates the recombination process¹². The 2 loxP sites are placed at the farthest 5’ and 3’ end of the target sequence respectively. Since we need to utilize the recombination activity and eliminate its self-splicing ability, the two loxP sites need to be compatible with itself but incompatible with each other. We chose lox511, lox5171 and lox2272 to pair with wild-type loxP sites^13,14, and examined their incompatibility with wild-type loxP.

We found that even though we’re putting Cre under a controllable promoter, its small leakage already can initiate self-splicing between 2 wildtype loxP (see Results for elaboration). This is undesirable as uncontrollable recombination could greatly damage the confidentiality of our result. The desired sequence could be recombined and expressed for a time and then be gone with the ongoing recombination. Our modelling result also shows that Cre expression needs to stay at a low level for a higher recombination rate.

To bring Cre expression under more stringent control, we made several different versions of Cre.

Firstly, we mutated some of the encoding codons to rare codons in hope that this would bring difficulty to translation and thus bring down the expression level.

Then, we added different degradation tags following it. We designed the tags utilizing the endogenous degradation system of E. coli¹⁵. We tested 5 tags, (YA)LAA, LVA, LAV, LVV, and (WV)LAA based on research literatures^16,17. With the support of our Modelling, we found that XXX tag best suits our need, with moderate steady state level and quick degradation dynamic.

As an alternative approach, we also tested the split-Cre construct¹⁸. Cre recombinase is split between its 59^th and 60^th amino acid, the N- and C-terminal fragments are attached to FRB and FKBP separately. When the inducer rapamycin is absent, the two fragments will not polymerize and no detectable Cre activity is found. After adding rapamycin into the culture, FKBP and FRB will polymerize, thus bringing the two Cre fragments into contact, and recombination activity will be gained.

EGFP is put under the ptetR promoter in place of Cre, and its fluorescence level is measured.

Cre is co-transformed with another plasmid containing a stably expressed mCherry with two wildtype loxP sites flanking. Afterwards the whole sequence is amplified through PCR, and if Cre is in the system, mCherry would be spliced from its plasmid, and the amplification result would be a short sequence, while the non-spliced mCherry would be fully amplified.

Cre is co-transformed with another plasmid containing a stably expressed mCherry flanking by two different loxP sites. The whole region is PCR amplified, and gel run to see if self-splicing event has occurred.

EGFP carrying all five tags have been cloned and measured for its fluorescent level.

Cre is co-transformed with two plasmids. One plasmid contains a stably expressed full-length mCherry flanked by 2 different loxP sites; the other carries a truncated version of mCherry, its flanking loxP sites match with the first plasmid. The truncated version is known to be inactive. If recombination occurs successfully, we would detect the first plasmid carrying the truncated version through PCR amplification and agarose gel analysis. Also, the fluorescent level would decrease.

We have constructed plasmids carrying chloramphenicol resistance gene with different stop-codon mutations. The gene is thus inactive, and acts as the target sequence. The two plasmids, carrying the target and other mutation necessary components respectively, are co-transformed. After incubation, the reverse transcriptase would be induced first, then Cre recombinase. The target sequence would be randomly mutated in the system, and if the mutation happens to successfully reverse the stop-codon back into its original coding sequence, the cell would grow successfully on solid medium containing chloramphenicol. By counting the number of cells grown, we could get a picture of the efficiency of single site mutation.

The surviving cells would be cultured and plasmid extracted. The plasmids would be sent for sequencing to prove that other sequences unrelative to the target is not mutated. It would also be retransformed into bacteria which has never encountered the system, and be planted on media containing chloramphenicol. If the cell could still grow normally, we could assume that the bacteria’s genome is not mutated in our mutagenesis process.

Since we’re using parts that are orthogonal of native bacterial systems, our system is expected to work independently in different species. The system is tested in ^{E. coli}, and will then expand to species including cyanobacteria and lactic acid bacteria.