Team:Hong Kong HKUST/riboregulator/characterization

To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter (BBa_J23102) to drive the expression of the cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible PBAD promoter (BBa_I0500) . The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source) (Figure 1. A). (fsd different between –TA and -CR).

For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock 1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system. After repression, the system needs to be activated when taRNA is expressed.

PBAD promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD. The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate pathway. The Pc promoter which is adjacent to the PBAD promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity of the PBAD promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can activate the PBAD promoter.