Part:BBa_K1442304:Experience

Applications of BBa_K1442304

The RNA promoters and terminators were designed to be tested both in e-coli and in human Huh 7.5 cells in order to determine their efficiency and to prove that the replicon system can function in human cells. Two dedicated testing modules were designed to that effect – one for mammalian (Part:BBa_K1442048) and one for bacterial cells (Part:BBa_K1442102). Both included a reversed GFP. The experimental setup requires the addition of a separate plasmid that carries the RdRP for bacterial cells and the addition of RdRP either in RNA or protein form for human cells. The two constructs used are shown below.

Due to time restrictions, we only managed to perform RNA promoter experiments in e-coli.

Experimetal Deasign

Test: GFP expression rate in cells co-transformed with plasmid that contains the promoter and a reversed GFP sequence and a plasmid containing the RdRP (Part:BBa_K1442100).

Positive Control: GFP expression rate in cells transformed with Biobrick plasmid that contains a gene for GFP with an inducible T7 promoter.

Negative Control: GFP expression rate in cells co-transformed with the plasmid that contains the promoter and a reversed GFP sequence and a plasmid containing a mutated version of the RdRP (Part:BBa_K1442101) that has been proven to be unable to produce successful replication of RNA.

Method

The characterisation of the parts was done using a 96-wells plate fluorescence reader – Tecan.

1. BL21 strain line cells were used to successfully co-transform the two plasmids containing the testing modules shown above. BL21 do not have any pre-existing antibiotic resistance and express the T7 polymerase gene. As many as 12 colonies which have both plasmids were overnighted in 5ml of LB containing the 2 antibiotics ensuring selectivity to make biological replicates. The RdRP plasmid has ampicillin resistance and the Promoters plasmid is resistant to chloramphenicol.

2. The following morning, 10 µl of the overnights were “refreshed” in 1 ml M9 media which unlike LB is not fluorescent but is less rich in nutrients. Again antibiotics were added accordingly.

3. Since the T7 promoter which governs the transcription of both plasmids is IPTG inducible, IPTG of 1mM concentration was added to the refreshed cell cultures.

4. The cells were left to grow and adjust to the new media for 6 hours.

5. The cells were re-refreshed when transferred to the 96 wells plate to be used in the Tecan. 10 µl of cells were added to 190 µl of M9 media plus ampicillin and chloramphenicol antibiotics and 1mM IPTG concentration. Two technical replicas for each biological were made. The experiment was conducted over 21 hours. The data obtained was a numerical value of growth by measuring optical density at 600nm (OD 600) and green fluorescent protein (GFP) taken at different wavelengths. To determine a value for the promoter strength, we averaged the GFP and OD values for all replicas. The GFP was then divided by the OD value to account for difference in growth across the samples due to the effect of the number of plasmids and IPTG.

Results

The results indicate that there was a very low level of GFP expression from both the test and the negative control compared to the positive control.

The figure above shows a comparison between the function of R5 RNA Promoter and the SD3 RNA Promoter. It shows that the SD3 is performing better than R5. However, it is still much less than the positive control expression. This might indicate that the functionality of the promoters is compromised, as the results of the mutant defective RdRP and the original RdRP are almost identical. The difference between the GFP levels between the 2 Promoters can be explained by the different sequences of the two parts. Assuming that an undiscovered RBS exists downstream of the reverse GFP which directs translation of the GFP independently from the RdRP, different efficiencies can be attributed. However, the mutated RdRP might be able able to bind to these alternative to the HCV sequences and indeed direct replication. Further investigation is needed to establish this.

Growth and GFP expression curves for both the RdRP and mutant RdRP carrying cells are very similar which indicates that there is no difference between the function of both and casts doubt over the efficiency of the RdRP.

Discussion and Further Work

A note to be taken is the extremely slow rate of growth of the cells containing 2 plasmids. This casts doubt over the health of the cells and their ability to support the plasmids, considering the strength of the T7 promoter. Issues about RdRP toxicity are raised as well. However, it is to be expected that cells which have been transformed with 2 plasmids instead of one experience slower growth rates. A necessary next step is to increase the time the experiments runs for and to ensure that the samples transferred to the plate reader have the same OD 600.

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