Team:Hong Kong HKUST/riboregulator/characterization
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<br><h2>P<sub>BAD</sub> Characterization<br> | <br><h2>P<sub>BAD</sub> Characterization<br> | ||
- | <b>(Please note that the following data has been shown to be problematic because our new data do not match our old data. We will update the following information as soon as possible)</b></h2><br> | + | <b>(Please note that the following data has been shown to be problematic because our new data do not match our old data. We will update the following information as soon as possible - HKUST iGEM 2015.23Jan2015)</b></h2><br> |
</div> | </div> | ||
Latest revision as of 03:38, 23 January 2015
Riboregulator Characterization
Introduction
Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system,
cis-repressing RNA, crRNA, contains a cis-repressing sequence which is located at the 5’ of the RBS and the gene of interest.
When the transcript is formed, the cis-repressing sequence can form a loop to form complementary base pairs with the RBS and blocking the ribosome's entry
to RBS. crRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. The taRNA is the component of the
system that
act as a key. 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.
Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate and “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 the iGEM 2006_Berkeley simply made variants of Lock 3 and Key 3. They put an alphabet at the end of the name every time they produced different variants 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 riboregulator variants from iGEM 2006_Berkeley. |
Riboregulator Results
![]() ![]() Figure 1. Fluorescence (F)/OD600 measurements of riboregulator pairs after arabinose induction and their corresponding controls.All samples were inoculated in M9 minimal salt medium overnight in no or various arabinose concentrations (%w/v). The samples were diluted around 10 fold the next day. Measurements were made when the samples reached around the mid-log phase (OD600 = 0.3 to 0.5). Graphs depict the triplicate mean + standard deviation. (A) Schematic diagram of the genetic context of the experiment. Note that the diagram generalized the CR and TA sequences. (B) Measurement for Lock 1 (BBa_J01010) and Key 1 (BBa_J01008) cognate pair. (C) Measurement for Lock 3(BBa_J01080) and Key 3 (BBa_J01086) cognate pair. (D) Measurement for Medium lock (BBa_K175031) and Key for medium lock (BBa_K175032) cognate pair. (E) Measurement for Lock 3c (BBa_J23031) and Key 3c (BBa_J23008).To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequences, trans-activating sequences and the RBS. The RBS sequence also had to be different for some of the riboregulator systems 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 so the interaction depends on the sequences. Since different teams used different RBS to design their cis-repressing sequences, 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 the 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 (Isaacs et al., 2004) (Figure 1. A). 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). Almost
full repression was observed for the three cognate pairs. 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). One possible reason could be that the RBS
sequence that we used for the controls of Lock 3c Key 3c was incorrect. For Lock3c the target RBS sequence was not mentioned. It seemed like a variation of
BBa_B0034 with shorter 3’ end. In order to build the construct type 2 and 3 (Figure 1. A), the RBS sequence had to be
deduced from the Lock 3c sequence. From the Lock 3c sequence, we have used a part of sequence that resembled the RBS (BBa_B0034
). The RBS sequence used may have been too short to be functional. Therefore, no fluorescence is observed when cis-repressing sequence is not present. On the other hand,
fluorescence can be observed when cis-repressing sequence is present because firstly, the RBS is sequence is correct, and secondly because the cis-repressing sequence failed
to repress the translation. After repression, the system needs to be activated when taRNA is expressed. After the addition of arabinose, taRNA is expressed. Out of the three cognate riboregulator pairs that were repressed, only two showed significant increase after arabinose induction. Lock 1- Key 1 cognate pair showed around 13-fold increase for both 1% and 2.5% (%w/v) arabinose induction. Lock 3- Key 3 cognate pair showed around 1.5 and 3 fold increase for 1% and 2.5% of arabinose induction respectively. Lock 1-Key 1 and Lock 3- Key 3 behaved differently for different concentrations of arabinose induction. Full induction was observed at 1% arabinose for Lock 1- Key 1 cognate pairs while full induction for Lock 3- Key 3 was observed at 2.5% arabinose. Statistically, no significant fold increase could be observed for Lock m- Key m cognate pair. |
Discussion
The fold increase for Lock 1- Key 1 is lower than that of riboregulator pair mentioned in the Isaacs et al.’s paper. |
Methods
Fluorometry |