Part:BBa_K315049:Design
Gene Spacer with tt
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The gene stuffer is made up of tetA fragment and a RFP fragment. The tetA fragment is made up of the first 855 base pairs of the tetA gene and the RFP fragment is composed of the first 336 base pairs of the RFP gene. The stuffer itself combines these two fragments and is 1191 bp long (the same size as the tetA gene). The stuffer gene theoretically lacks any transcriptional terminators as it is composed of initial gene sequences of the tetA and RFP. These intial sequences are highly unlikely to have any transcription terminators as the sequences would not succesfully code for their respective proteins if transcription terminators were present this early. The intial purpose of builiding the stuufer was to build the pLac+RBS+Stuffer+RBS+RFP gene sequence. This construct would allow us to further investigate the cause of the lower expression levels of reporters placed after tetA. If fluorescence data for this construct yields similar results to cells with the pLac+RBS+tetA+RBS+RFP insert, then we can conclude that the minimal fluorescence in both constructs is a result of the distance between the reporter (RFP) and the promoter (pLac). However, if fluorescence data resembles the results from cell types with the pLac+RBS+RFP+RBS+tetA insert, then the results would strengthen the premise that the tetA gene has a transcriptional terminator that is responsible for diminished fluorescence. The concept behind the stuffer can be used to test other genes that are suspected to have transcription terminators. Additionally, the stuffer can be used as a spacer gene to separate different sequences. A spacer might be of use to lower the strength of a promoter and thus may help in manipulating the signal of a gene circuit.
Source
The gene stuffer is made up of tetA fragment and a RFP fragment. The tetA fragment is made up of the first 855 base pairs of the tetA gene and the RFP fragment is composed of the first 336 base pairs of the RFP gene.