Part:BBa_J15001

strong synthetic E. coli ribosome binding site with SacI site.

This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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 Strong". We also characterised RBS mediumand RBS weak.

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 strong RBS characterised here (labeled Str above) showed very weak activity, giving a signal strength of the same values as 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 were contradictory to what we expected, with the RBS effectively terminating all expression of sfGFP in the cell, since it showed the same values as the negative control. There are possible explanations, but our sequencing showed no errors in the plasmid produced and used, and no other errors were noted. The other two RBS tested also showed lower values, but did show some expression compared to the negative control. We invite other iGEM teams to also characterise this part in order to further determine the nature of this RBS.