Improvements on BBa_K592023 and BBa_K592024
(Blue Fluorescent Protein mTagBFP generators)
The 2011 Uppsala Sweden iGEM team had previously submitted characterization data for parts BBa_K592023 and BBa_K592024. However, we discovered that they did not put a terminator after the mTagBFP CDS. We wondered if the observed fluorescence outputs will be different with versus without a terminator. Therefore, we decided to perform an experiment for comparison. The experimental results showed that the observed fluorescence levels were lower without a terminator.
Given our results, we would like to caution future users in referencing the relative promoter strengths reported by Uppsala Sweden 2011, as their measured expression levels, generated by parts without a terminator, may not fully represent the true strength of the tested promoters.
THE INVESTIGATION
We hypothesized that the expression level of mTagBFP is affected by the presence or absence of a terminator.
An experimental construct was built where the mTagBFP generator (BBa_K592100) was driven by a constitutive promoter (BBa_J23101) and a medium RBS (BBa_B0032). It was compared against its counterpart which harbored a terminator, BBa_B1006 3’ to the CDS. Constructs without promoters served as controls for auto-fluorescence. We then performed the same experiment but used BBa_B0034 as the RBS instead. Both of the results showed that the observed blue fluorescence outputs were lower when the transcription was not properly terminated.
![](https://static.igem.org/mediawiki/2016/4/48/Team--Hong_Kong_HKUST--Parts_Improvement.png)
Our results indicated that the proper expression of mTagBFP requires a terminator; thus the previously submitted part BBa_K592023 (BBa_B0032-BBa_K592100) and BBa_K592024 (BBa_B0032-BBa_K592100) are translational units only and should not be considered as acceptable alternatives to mTagBFP generators, which have terminators.
In this experiment, we have improved BBa_K592023 (BBa_B0032-BBa_K592100) and BBa_K592024 (BBa_B0034-BBa_K592100) by adding a terminator BBa_B1006 3’ to these parts. Both of the improved parts, BBa_K1899001 (BBa_K592023-BBa_B1006) and BBa_K1899002 (BBa_K592024-BBa_B1006) were submitted to the Parts Registry this year. With the improved parts, we hope to provide future users with better reporters for their assays.
*For a detailed documentation of this characterization and the materials or methods used, please refer to our PDF version of this investigation.