Team:Tokyo-Nokogen/Project/Counter

RTC counter - signal counting switch
[Introduction]
Gene engineering has enabled us to cut and connect the genes that we want to control. By this technique, we can design the genes and control their expression. The developments in synthetic biology offer so many attractive approaches for the regulation of gene expression. One of these approaches, the riboregulator using signal switch has been reported [1] [2]. This system can control the protein’s expression amount by adding the signal, and can express another protein by lag time. We focused on this counter signal system, attempting to apply it to our ESCAPES system. Our final goal is to design a signal counting switch to simplify the processes in view of automation. This would allow us to use only one signal (e.g., red light) in our team’s ESCAPES system instead of using a different signal for each step. We focus on riboregulated transcriptional cascade (RTC) counter, which is based on a transcriptional regulation.

he concept is illustrated in Fig. 1 for the two target genes A and B. A constitutive promoter drives transcription of Gene A and T7 RNA polymerase (RNAP), whose protein transcribes gene B, which is regulated by a T7 promoter. However, the translation of all these genes is controlled by riboregulators, whose cis and trans elements silence and activate posttranscriptional gene expression, respectively. A cis-repressor sequence (cr), located between the transcription start site and the ribosome-binding site (RBS), is complementary to the RBS. This complementarity causes it to from a stem-loop structure upon transcription and prevents binding of the 30S ribosomal subunit to the RBS, thus inhibiting translation. A short, transactivating noncoding RNA (taRNA), driven by an inducible promoter, binds to the cis repressor in trans, thus relieving RBS repression and allowing translation. With this riboregulation, each node (i.e., gene) in the cascade requires both independent transcription and translation for protein expression (Fig. 2). This cascade is able to count frequency of the signal by expressing a different protein in response to each time the signal is applied.

This RTC counter system is used for ESCAPES system by connecting color light switch and aggregation and lysis parts. First, we constructed the model system using this RTC counter. Fig. 3 and Fig. 4 show the model system. These model parts will work by cotransforming them and using arabinose induction. By this induction, taRNA of model part is transcribed and this transcription product binds to crRBS, resulting in RFP expression. After that, T7 RNA polymerase is expressed and binds to T7 promoter. And then, GFP is expressed after a lag time.

[Methods]
(1) Preparation of crRBS
We ordered the sense strand and antisense strand of crRBS having XbaI restriction site on 5’ terminus region and SpeI restriction site on 3’ terminus region. We prepared dsDNA from these strands by heat process (95℃ for 5 min.→slope for 15 min.→80℃ for 1 min→slope for 20 min.→70℃ for 1 min →slope for 20 min.→60℃ for 1 min→slope for 15 min→50℃ for 1 sec.→slope for 20 min.→25℃). Then the dsDNA was digested with XbaI and Spe, and ligated in a single tube with BioBrick plasmid backbone digested with XbaI and Spe (Fig. 5).

(2) Construction of new part; RTC counter element 1 and 2
We have already constructed the model parts [PBAD-RBS-taRNA-Terminator-high promoter-crRBS-RFP-Terminator] and [High promoter-crRBS-T7RNAP-Terminator-T7pro-crRBS-GFP-Terminator] following the basic BioBrick assembly procedures. We planned to test our model system (Fig. 4) by transforming Escherichia coli DH5a with the two constructed plasmids and pulse the transformed cells twice with the inducer arabinose and measuring for 5 hours the fluorescence of RFP and GFP at 607 nm and 511 nm, respectively. The absorbance at 607 nm would be expected to increase after the first pulse and the absorbance at 511 nm would increase after the second pulse. Unfortunately, we could not complete our model system and were therefore unable to confirm whether they function as expected.

[Reference]
[1] Ari E. Friedland, et al., Science 324, 1199 (2009). [2] F. J. Isaacs et al., Nat. Biotechnol. 22, 841 (2004).