Part:BBa_K3252027
Very Weak Ribosome Binding Site BBa_B0033
Parts BBa_K3252027 is very weak RBS BBa_B0033. This RBS was used to express LuxN protein. This part was assembled with LuxN gene (BBa_K3252030) and BBa_B0015 transcription terminator by ordering custom gene synthetic.
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]
Characterisation by Team:Chalmers-Gothenburg 2021
Aim
Characterisation of several ribosome binding sites in order to expand upon the options and knowledge available to future iGEM teams. Of the parts characterised, this one was labeled "RBS Weak". We also characterised RBS mediumand RBS Strong.
Experimental design
Due to the short length of RBS, we decided to utilise a modified PCR primer method, whereby the sfGFP-containing plasmid pYTK001 from the MoClo kit was amplified by a forward and reverse primer pair containing an overhang with the RBS, designed as that it blocked and overwrote the natural RBS present, replacing the native RBS.
Method
pYTK001 was mixed with a custom forward/reverse primer pair and PCR reaction carried out according to protocol using Phusion polymerase. The produced solution was purified using gel purification followed by gel purification kit from ThermoFischer, and the resultant linear fragment was cyclised using Gibson assembly protocols, followed by transformation into competent E.coli strain DH5-alpha. The plated cells were inoculated, grown overnight and plasmids extracted using ThermoFischer plasmid miniprep kit and protocol. From the extracted plasmids a sample was sent to sequencing, while the remainder was used to transform again, and following overnight growth, inoculated again, followed by equalizing the OD of each sample. Alongside a positive control consisting of unmodified pYTTK001 sfGFP-containing plasmid, a negative control of DH5-alpha E.coli containing plasmid pYTK002 without GFP and a blank of LB media, the fluorescence was measured with three replicates of each RBS using a plate reader and the strength of each RBS was measured.
Results
The data obtained was collected and analysed, giving the following result:
Results of the three RBS characterised by team Chalmers/Gothenburg 2021.
As can be seen, the weak RBS characterised here (labeled Weak above) showed very weak activity, giving a signal strength marginally above the negative control. The positive control of base pYTK001 with sfGFP gave a very strong signal, magnitudes greater than the rest. The sequencing also showed no mutations or errors in neither the RBS site nor the sfGFP sequence.
Discussion
The results for this part were satisfactory, showing a weak expression level as expected, just above that of the negative control. Our sequencing additionally showed no errors in the plasmid produced and used, and no other errors were noted. The other two RBS tested showed lower values, with the strong RBS showing the same values as the negative control control. We invite other iGEM teams to also characterise this part in order to further determine the nature of this RBS, particularly in light of the weak results of our other tests.
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