We make helpful contributions to future teams by introducing a new part type---dtRNA, to the iGEM community. Degradation-tuning RNAs allows for enhancing protein expressions without posting an extra metabolic burden to the host, modulating the dynamics of genetic circuits and regulating non-coding RNA functionalities by modulating degradation rates. On the other hand, the small size of dtRNAs saves much effort in the molecular cloning process. We tested the functionalities of several dtRNA structures in the context of different promoters, ribosome binding sites, and coding sequences to confirm their versatility for integration into other circuits. The standardized biological parts form of dtRNAs would facilitate its integration into existing gene circuits and supports the future construction of novel biomolecular reaction networks with tunable parameters and increased complexity for other teams.
On the other hand, we have also documented some new data on the following existing parts: BBa_E0240, BBa_E0030
These parts are fluorescent protein generator devices containing an RBS and a downstream fluorescent protein like GFP or EYFP. Since they could stand as one of the building materials for higher-order devices and circuits and are frequently used in iGEM kit plates, a well-documented characterization would help to provide more information about these parts for future use by other teams.
The most probable way for usage of BBa_E0240 is to integrate various promoters upstream of the RBS and use GFP as reporters to measure expression, so we add three constitutive promoters with different strengths (J23106, J23109, J23116) upstream of the RBS and compare the bacterial fluorescence to test the performance and universality of this device.
The samples for measurement are prepared as follows:
1.Use inverse PCR to obtain the vector fragment, and the position of the joint is right upstream of the RBS B0032.
2.Commercially purchased single-stranded DNA integration fragments(promoter sequences) with two homologous arms are inserted into the linearized vector through HiFi assembly. (Approximately 50:1 in molar ratio)
3.E.coli DH5α competent cells are transformed with finished HiFi assembly reactions, and colonies are picked and sequenced on the next day.
4.Colonies with correct sequences are cultured for 8h and then transferred to a microplate with 100-fold dilution, GFP fluorescence (excitation=488 nm, emission=515 nm) and OD600 are measured every 15 minutes for 16 h duration.
The results are as follows:
Figure.1 Fluorescence curve of BBa_E0240 under the control of different promoters
From this graph, we clearly see that the promoter strength order is J23106, J23116, and J23109, from high to low. The qualitative analysis accord with previous reports, but the comparison of quantitative promoter strength should be compared to ensure the validity of this part, and we take the fluorescence peak value as a measuring standard for promoter strength.
Figure.2 Comparasion of promoter strength
As the graph shows, we found good correlations between previously reported promoter strengths and our data, some still observed deviations could be attributed to the difference in measurement procedures or data processing. Anyhow, the result indicates this part might be a good material for constructing genetic circuits.
Figure.3 Growth curve of BBa_E0240 under the control of different promoters
Since the vector of this part is a high-copy plasmid(pSB1A2), we also collected the growth curve of these transformants to test the metabolic burden of this part under the control of different promoters. The result shows that the bacteria growth is barely affected, and all three transformants' density reaches their plateau within 10 hours with a slight yet observable difference between medium strength J23106 and the rest two. This should raise caution for using high-strength promoters such as J23100 as it might introduce high metabolic pressure.
Next, we characterized BBa_E0030 for measuring its fluorescent curve under the control of J23106 and B0032, and compare it with GFP(BBa_E0030), to test the effect of the reporter gene on measurements.
Figure.4 Fluorescent curve of BBa_E0030 under the control of J23106 and B0032
Figure.5 Growth curve of BBa_E0030 under the control of J23106 and B0032
The result shows that EYFP and GFP hold different dynamics for fluorescence growth, partially due to the maturation and quenching of fluorescent proteins and different background signals. Yet, their growth curve is roughly the same. These results also emphasize the need for choosing appropriate reporter genes in measurement experiments.