Team:Hong Kong HKUST/Experiments
General Protocols
With regards to wet lab work, several experiments have been carried out to prove the functionality of our Biscuit.
We provide you with a list of general protocols we have followed while conducting our experiments, so that future iGEM teams can replicate them too:
link to our general protocols
RNA In Vitro Transcription (IVT) and interaction assay
Aim:
To assess whether the sgRNA and asRNA can be transcribed successfully and hybridized in vivo, an in vitro assay was designed to assess the transcription of the RNAs and the feasibility for the RNAs to hybridize in vitro.
Here are all the protocols we used for the RNA IVT and interaction assay
link to our RNA IVT and interaction assays protocols
Flow of experiment:
sgRNA and asRNA is synthesized via in vitro transcription (NEB HiScribe T7 in vitro transcription kit). Part of the transcript is mixed together to allow for hybridization. The part is then analyzed on both formaldehyde denaturing agarose gel and non-denaturing agarose gel to assess transcript size and hybridization respectively. (figure 1)
| Denaturing Gel (formaldehyde) | Non-denaturing Gel |
|---|---|
| Destroys secondary structure | Preserves secondary structure |
| Assess actual size of transcript | Assess interaction |
Step 1 - PCR synthesis of IVT templates
The 2 RNA’s transcription templates, designed according to NEB IVT kit manual, were created by using PCR to insert a T7 promoter in front of the transcription template using an overhang forward primer. (Figure 2)
Thereafter, the PCR product is run through an agarose gel to separate impurities and inspect the sizes of the IVT templates before undergoing gel purification.
Results:
The size of IVT template is as expected to be around 150bp, with sgRNA expected to be 155bp and asRNA expected to be 157bp. This proves the IVT template sizes are correct and can be purified for IVT reaction. (Figure 3)
Step 2 - In vitro transcription (IVT) using NEB T7 HiScribe Transcription Kit
To synthsize the sgRNA and asRNA, 75ng of gel purified DNA transcription template was added into the IVT reaction mixture. The reaction is set for 16 hours of incubation at 37°C, using a thermal cycler to prevent evaporation.
The transcripted RNAs is treated with DNase1, then precipitated by sodium acetate / ethanol.
The sizes of the 2 precipitated RNAs are confirmed by using denaturing formaldehyde-MOPS 2% agarose gel. Since sgRNA is known to form multiple secondary structures, heat denaturation at 65 °C for 7 minutes is needed prior to loading into the gel to remove all secondary structures,allowing for accurate assessment of its size.
Results:
The gel photo above indicates the successful transcription of the sgRNA and asRNA, with the size suspected to match expected size (sgRNA=138bp, asRNA=140bp).
Step 3 - RNA in vitro hybridization and visualization
In order to test the environment needed for in vitro hybridization, 4 different environments were tested in combination of using 3x Hybridization Buffer (HB) and the use of initial denaturation. Regardless of the conditions being tested, the 2 RNAs are incubated together at 37°C for 45 minutes, before snap cooling to 0°C to preserve the hybridized structure and prevent RNase degradation. (protocol we used for the experiment)
It is known that salt content is crucial to RNA duplex formation [1], thus, 3x Hybridization Buffer is used to provide the necessary salt content (60mM K+ and 0.2mM Mg2+) and stabilize the RNAs. DEPC water is used as a negative control to the testing of hybridization buffer.
Since this assay is designed as an in vitro demonstration of the in vivo hybridization, the temperature of incubation is set to 37°C to simulate the in vivo environment with no initial denaturation. As a fail-safe test, the necessity for initial denaturation is also tested as the in vitro hybridization is expected to be less efficient without the Hfq Chaperone. Therefore, denaturing the RNAs before slow cooling will remove the RNAs secondary structure while promoting the complementation and hybridization during slow cooling.
A non-denaturing 2% MOPS agarose gel is then used to visualize the hybridization.
Results:
Index
| HB/D | Hybridization Buffer + denature + slow cool* + 37°C incubation |
| HB/37 | Hybridization Buffer + 37°C incubation |
| D/D | DEPC H2O + denature + slow cool* +37°C incubation |
| D/37 | DEPC H2O + 37°C incubation |
| L | Ambion Century™ - Plus RNA Markers |
| sg | sgRNA BBa_K3017061 (138bp) |
| as | asRNA BBa_K3017060(140bp) |
As shown in the above gel photo (Figure 5), only lane AB/37 shows an extra band above the thick band at the bottom. This band indicates the hybridization of sgRNA and asRNA and is therefore running slower in agarose gel.
This result shows that salt content provided by the hybridization buffer (60mM KCl, 6mM HEPES, 0.2mM MgCl2 at 1x), which is similar to that of a cellular salinity [2], is crucial to the hybridization. This result also indicates that the RNAs are able to hybridize with each other in 37°C incubation with no initial denaturation needed, which is identical to cellular environment. These results suggest that the RNAs may be capable of hybridizing in vivo. With endogenous expression of Hfq protein in E.coli, the asRNA will be stabilized due to Hfq protein binding to pot 42 and attract other ssRNAs to hybridize.
Thus, we suspect the in vivo hybridization efficiency would be higher than our results.
Transformation and expression of dCas9 in DH5-α E.coli
Aim:
As one of the most important components of our circuit, dCas9 needs to be cloned and expressed in the cells. The dCas9 sequence used was kindly given by Dr. Ho Yi Mak, a professor for Life Sciences in HKUST, who has experience working with dCas9. However, the sequence of that dCas9 was noticed to have 5 PstI cut sites within the coding sequence. This meant that the standard assembly method using EcorI, XbaI, SpeI and PstI would not be compatible.
Flow of experiment
We replaced the PstI cut site by adding a SalI cut site after the PstI site by PCR. The other basic parts (BBa_K608006 and BBa_B0015) that were used in subcloning were also added with the SalI cut site. The three parts were then constructed in a BBa_PSB1C3 vector to form a dCas9 expression vector. The resulting plasmid was then transformed into DH5α E.coli using heat shock, and the cells were selected by chloramphenicol followed by Miniprep plasmid extraction and PCR verification (Figure. 6).
Here are all the protocols we used for the dCas9 expression check assay
link to our dCas9 expression protocols
Following transformation, we further went on checking the expression of dCas9. Cell lysis was prepared by sonication in NP-40 lysis buffer. The protein concentration of the lysis was checked by Bradford Assay against the BSA standard curve (Figure. 7). 10ul of cell lysis was then used to run through the SDS-PAGE (5% stacking, 6% resolving).
After the electrophoresis, the gel was stained with Coomassie Blue to visualize the protein (Figure. 8). As shown in the gel, there is an extra band much larger than 82kDa which exists only in the dCas9 transformed cells, but not appear in the cells transformed with an empty vector. This result showed the first clue of the expression of dCas9 whose molecular weight is expected to be 160kDa (Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) | Abcam).
To further verify whether the extra band was dCas9, we replicated the experiment but used a Western Blot targeting the 6XHis tag at the C-terminal of dCas9 as opposed to visualizing using Coomassie Blue. The Western Blotting was done using a His-probe antibody (1:2000, Rabbit) and a secondary HRP-goat anti rabbit antibody (1:2000). 500ul of ECL detection reagents (Amersham) was then added, and the band was visualized using light-sensitive X-ray films.
Figure 9 shows a clear band that only appears in the dCas9 transformed cells, verifying that the extra band shown in figure 8 was indeed the dCas9.
Characterization of bistable switch components
sgRNA-dCas9 suppression assay
Aim:
This construct is to test if sgRNA works in vivo to form a complex with dCas9, and suppress fluorescent protein levels. It also aims to provide a baseline of fluorescence and suppressed fluorescence level. As dCas9 is a large protein (1375 AA), we suspect that co-expressing a fluorescent protein(FP) with dCas9 may be substantial metabolic stress to the cell, thus lowering the expression level.
Design:
This construct consists of an sgRNA for GFP expressed under a pBAD promoter and constitutively expressed GFP (K3017066). A constitutively expressed dCas9 expression construct (BBa_K3017069) will be subcloned into the dCas9 control plasmid using standard assembly.
By using an inducible promoter, we can compare the GFP level before and after turning on the arabinose-inducible pBAD promoter, thus evaluating the suppression effect caused by CRISPRi (brought about by the dCas9-sgRNA complex). The unsuppressed GFP level (before arabinose induction) also gives us a figure of how the GFP expression level would be affected when it is co-expressed with a dCas9 protein.
Proposed Characterization of asRNA depression and crosstalk between components
Aim:
This is a proposed characterization, designed to test whether expressing the paried asRNA can in fact derepress the repression effect induced by the sgRNA in vivo.
Design:
The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein (GFP or RFP), asRNA under pBAD promoter, and sgRNA (GFP). Under the regulation of pBAD promoter, asRNA is transcripted when arabinose is added to the culture medium. The transcription start site of the pBAD promoter has been identified according to Brzozowska et al. (2018) [3], in which the asRNA has been placed on the Transcription Start Site (TSS). For all sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used to allow for faster response rate between repressed state to derepressed state after arabinose induction. The different combinations of fluorescence protein and sgRNA allow us to characterize different behavior of the asRNA as in the following diagrams and table.
| Construct | Behavior of Characterizing |
|---|---|
| GFP_sgRNA(GFP)_asRNA | asRNA ability to derepress a CRISPRi effect under arabinose induction with pBAD |
| GFP_sgRNA(RFP)_asRNA | sgRNA mismatch with different FP |
| RFP_sgRNA(RFP)_asRNA | asRNA mismatch with sgRNA |
Old Part Characterisation
Aim:
Characterize the LacI-regulated promoter (BBa_R0010) used in our circuit under different pH conditions.
Design:
The bistable switch uses the LacI-regulated promoter as one of the two promoters. Glucose causes repression of the promoter. We believe that in different pH, glucose is modified thus affecting its repression capabilities. In order to understand the degree of control over its activity in different environment conditions, we decided to characterise promoter activity induced by glucose in different pH 5-9[4]. When this promoter is coupled with GFP, the resulting GFP expression indicates the activity.
Flow of Experiment:
BBa_K741002 (pLac-RBS-GFP-T construct) containing BBa_E0040 GFP is used in the experiment. Cells transformed with this part are mixed into LB of pH range 5-9. 0.1M Glucose is then added to them. A control without the addition of glucose is also included in this experiment. There is a leaked expression of pLac observed in this construct. The cell samples with and without glucose inducer are treated in the LB for same duration. The OD and fluorescence of these samples are then measured using a plate reader.
Link to our old part characterisation protocol
Results:
pLac on its own shows leak expression while that with added glucose shows repressed activity. On plotting a graph of fluorescence/OD against pH, it was observed that the overall trend of GFP expression decreases with an increase in pH. This can be visualized in the graph of repression (difference between leak expression and glucose-induced expression) against pH.
Results are visualized in the pictures below.
Our numerical results are tabulated as follows:
References:
[1]Tan, Z. J., & Chen, S. J. (2006). Nucleic acid helix stability: effects of salt concentration, cation valence and size, and chain length. Biophysical journal, 90(4), 1175–1190. doi:10.1529/biophysj.105.070904
[2]SCHULTZ, S. G., & SOLOMON, A. K. (1961). Cation transport in Escherichia coli. I. Intracellular Na and K concentrations and net cation movement. The Journal of general physiology, 45(2), 355–369. doi:10.1085/jgp.45.2.355
[3]: Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor
Natalia Brzozowska, Jane Gourlay, Ailish O’Sullivan, Frazer Buchanan, Ross Hannah, Alison Stewart, Hannah Taylor, Reuben Docea, Greig McLay, Ambra Giuliano, James Provan, Katherine Baker, Jumai Abioye, Julien Reboud, Sean Colloms
bioRxiv 290155; doi: https://doi.org/10.1101/290155
[4]Politi, N., Pasotti, L., Zucca, S., Casanova, M., Micoli, G., Cusella De Angelis, M. and Magni, P. (2019). Half-life measurements of chemical inducers for recombinant gene expression.