Team:Hong Kong HKUST/riboregulator/characterization
From 2014.igem.org
Riboregulator Characterization
Introduction
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.
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
Introduction
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 (Schleif, 2010). |
PBAD promoter BBa_I0500 in Part Registry
There are several PBAD promoters in the Part Registry. The promoter that we were interested was BBa_I0500 because of two reasons. First, BBa_I0500, along with PBAD, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of PBAD can only work on strain that are AraC+. By coupling the araC gene with the PBAD promoter, we can be free from such restraints. Second, BBa_I0500 needed debugging. BBa_I0500, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter responded to the arabinose induction. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge results show that it was an “on-or-off” response. We wanted to analyze these problems so that the part could be more reliable for other users. Cambridge cited a paper that mentioned that variation in response could have resulted from cell strain variation Khelbnikov, 2001) |
Results
All-or-none response observed for individual cells. ![]() Figure 1. Forward scatter intensity (FSC) versus GFP graphs for samples with PBAD promoter regulating GFP generator.All samples were inoculated in M9 minimal salt medium overnight in various arabinose concentrations (%w/v). The samples were diluted around 10 fold the next day. Sample were fixed and the fluorescence was measured using flow cytometer. The graphs were plotted for the control constructs, pSB3K3-BBa_E0240 (-) and pSB3K3-BBa_I20260 (BBa_J23101) in the absence of arabinose. FSC versus GFP graphs for pSB3K3-BBa_I0500-BBa_E0240(BBa_I0500) in 0, 0.2 and 1.0% arabinose concentration were plotted. Each set of graphs were obtained for three different cell strains, DH10B, DH5α and BW25113.The distribution can also give us some idea of PBAD promoter leakage in different cells strain. If the data points are on or near the boundary between Q3 and Q4, we know that the promoter is leaky. The results indicate that the promoter is relatively more leaky in DH10B compared to other cell strains. ![]() Figure 2. The percentage of cells in induced and uninduced state, and RPU across different arabinose concentration.Q3 and Q4 represent the 3rd and 4th quadrants of the forward scatter versus GFP curve mentioned in Figure 2. The experimental condition was same as the procedure mentioned in the caption of the Figure 1. The left y-axis is for the percent of cells in Q3 and Q4 while the right y-axis is for RPU. Graphs depict the triplicate mean ± standard deviation. (A) Graph for pSB3K3-BBa_I0500-BBa_E0240 in DH10B. (B) Graph for pSBK3K3-BBa_I0500-BBa_E0240 in DH5α. (C) Graph for pSB3K3-BBa_I0500-BBa_E0240 in BW25113.
RPU of PBAD in different cell strains ![]() Figure 3. RPU of PBAD promoter in three different cell strains across different arabinose concentration.Relative Promoter Unit of PBAD promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using flow cytometer. The graph represent triplicate mean ±SD.
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by other experimental factors such as genetic context. Because RPU is a ratio to fluorescence measurement, the effects caused by these factors can be minimized (See Methods for RPU calculation). PBAD Leakage 3-D graphs for DH10B and DH5α Figure 4. Fluorescence and OD600 measurements of DH10B and DH5α induced in different arabinose concentrations.Triplicate of DH10B and DH5α samples were inoculated in deep well 96 well plate overnight in M9 minimal salt medium. Arabinose was added to match the final working concentration from 0 to 1.0 % (w/v) with 0.2% increments. Fluorescence and OD600 was measured every two hours for ten hours. (A) Increase of OD600 measurement for DH10B strain in different arabinose concentration. The graph represents triplicate mean ± SD (B) Increase of OD600 measurement for DH5α strain in different arabinose concentration. (C) Fluorescence VS arabinose concentration VS Time 3-D graph for DH10B. Each point represent triplicate mean. (D) Fluorescence VS arabinose concentration VS Time 3-D graph for DH5α. Each point represent triplicate mean.
The OD600 value reflects cell concentration. OD600 for both strain in different arabinose concentration increased exponentially. Growth rate for cells without arabinose induction (0%) was greatest for both DH10B and DH5α. For other concentration of arabinose induction the growth rates were similar for both of the cell strains (Figure 4. A and B). The similar growth rate across the 10 hour period indicates that the cell concentration for samples in different arabinose concentration increased similarly and therefore the cell concentration at each point of measurement was similar across different arabinose concentration. We can therefore assume that differences in the cell concentrations have minimal effect on the different fluorescence levels at different arabinose concentration. |
Discussion
Groningen 2011 results may not truly represent the gradual induction of PBAD promoter |
Methods
Construction Measurement
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Schleif R. AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev (2010) 1–18. |