Part:BBa_K4511007

Reporter device with J23106 , B0032, and GFP for dtRNA1 strength measurement

This part is a measurement device for degradation-tuning RNAs, which contains medium strength J23106 promoter, medium RBS B0032, and a reporter protein coding sequence GFP. The effectiveness of dtRNA is evaluated by measuring GFP fluorescence and bacterial growth curve. It could be used as an inter-lab study device to compare experimental conditions among different labs. This plasmid could also be directly transformed into other species or strains of E.coli to study the effectiveness of dtRNA on another chassis.

Characterization by 2022 team HUS_United

This year our team wishes to introduce a new kind of basic parts-degradation tuning RNAs to the iGEM community. The dtRNAs are reported to be capable of modulating mRNA degradation rates by resisting or facilitating endogenous RNase digestion. We plan to add this compact(10-60 bp) component upstream of the RBS B0032 in BBa J364009, in order to enhance GFP reporter signals without introducing metabolic burden to the host organism. 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:GFP fluorescent curve before/after adding dtRNAs

Figure 2:Growth curve before/after adding dtRNAs

We clearly see that introduction of dtRNA1 enhanced GFP fluorescence to nearly 3 folds, but the growth curve remains nearly unchanged. Therefore, we successfully improve this part by adding a small RNA structure in front of its RBS. The integrated version could be further used for inter-lab studies or measurement of dtRNA strength in different labs or contexts.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 753