Introduction

Last year our team has proposed a SynTF-SynPro approach which enables others to construct a customized and orthogonal transcriptional network in mammalian cells. The structures of the corresponding silencing- or activating-form of SynPros were pSV40-N*RE and N*RE-minCMV, in which N*RE is short for responsive elements of N repeats.

This year we took it a step further to create our engineered transmembrane binary logic gates in eukaryotic cells based on the foundational concepts we have constructed upon last year. We made use of the SynTF-SynPro system to function as our Combiner, which executes the final computation of our genetic circuits. To improve the signal-to-noise ratio of our Combiner and to allow others to more easily use our toolbox, we attempted to optimize the structure of the SynPros and constructed N*RE-CMV, which replaces the pSV40-N*RE to respond to transcriptional repressors. Furthermore, we also conducted experiments to characterize our N*RE-CMV, ensuring that it performs exceedingly better than the previous pSV40-N*RE. We have substituted all the pSV40-N*RE with N*RE-CMV when wiring our genetic circuits and picked 8*ZF21.16-CMV for specific demonstration of our parts improvement. For more details, refer to the existing BioBrick page or the improved BioBrick page.

Place of responsive elements

We have experimentally discovered that the place of N*RE can have an impact on basal expression of SynPros. Thus, when designing transcription repressing circuit, we hope that the addition of the N*RE will not bother the original expression of the promotor. Unfortunately, we realized that when the N*RE is placed downstream the promotor, the basal expression of the promotor is effected without the transcriptional repressor. Therefore, we made an attempt to adjust the position of N*RE to upstream of the promotor. Interestingly, the basal expression of the promotor is less affected as compared to the previous design. Actually, similar phenomenon have been reported previously that reporter activity was highly variable among the four different TALE binding sites introduced between the promoter and the reporter coding sequence while the introduction of multiple copies of each of the four different operators upstream from the promoter caused only minimal variability in reporter expression as suggested by Gaber R, et al, 2014 (PMID: 24413461).

pSV40 vs pCMV

To better verify that the transcription repressor can effectively repress the expression of its responsive circuit, we attempted to increase the basal expression for a more precise comparison. As a result, we designed a simple genetic circuit in which a EGFP-P2A was put downstream the promotor to test and compare the basal expression of pSV40 and pCMV. We used fluorescence microscopy to detect the expression of the reporter gene 24 hours after transient transfecting 293T cells with the two mentioned circuits, respectively. We found that pCMV has a significantly higher basal expression than that of pSV40, providing us a more ideal alternation to construct our transcriptional repressor-based Combiner circuit.

Figure 1. Flow cytometry data of previously designed SynPros based on pSV40. It can be clearly identified that the basal expression of pSV40 is easily effected by the adding of responsive elements. (sTF is short for silencing-form transcriptional factors.)

Figure 2. Flow cytometry data of the improved design of SynPros based on pCMV. It is shown that the basal expression of pCMV is barely effected by the adding of responsive elements. (sTF is short for silencing-form transcriptional factors.)

Figure 3. Fluorescence intensity comparison of pCMV-EGFP-P2A and pSV40-EGFP-P2A 24 hours after transfection. The expression level of pCMV is significantly higher than pSV40.