Team:Hong Kong HKUST/riboregulator/characterization


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Riboregulator Characterization


Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest. When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team.
Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:

Name and registry code Group Cognate pair
Lock 1 (BBa_J01010) Key 1 (BBa_J01008) iGEM 2005_Berkeley (Golden Bear) Yes
Lock 3 (BBa_J01080) Key3 (BBa_J01086) iGEM 2005_Berkeley (Golden Bear) Yes
Medium lock (BBa_K175031)Key for medium lock (BBa_K175032) iGEM09_TUDelft Yes
Lock 1 (BBa_J01010) Key 1 (BBa_J01008) iGEM 2005_Berkeley (Golden Bear) Yes
Lock 1 (BBa_J01010) Key 1 (BBa_J01008) iGEM 2005_Berkeley (Golden Bear) Yes
Lock 1 (BBa_J01010) Key 1 (BBa_J01008) iGEM 2005_Berkeley (Golden Bear) Yes

Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.

Riboregulator Results

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 Characterization


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.

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