iGEM 2019 — Team Hong_Kong_HKUST

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# Team:Hong Kong HKUST/Notebook

Team:Hong Kong HKUST - 2019.igem.org

[![](https://static.igem.org/mediawiki/2019/9/99/T--Hong_Kong_HKUST--biscuitlogo.png)](https://2019.igem.org/Team%3AHong_Kong_HKUST)

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Project

[Description](https://2019.igem.org/Team%3AHong_Kong_HKUST/Description)
[Experiments](https://2019.igem.org/Team%3AHong_Kong_HKUST/Experiments)
[Application](https://2019.igem.org/Team%3AHong_Kong_HKUST/Application)
[Safety](https://2019.igem.org/Team%3AHong_Kong_HKUST/Safety)
[Logbook](https://2019.igem.org/Team%3AHong_Kong_HKUST/Logbook)

[Modelling](https://2019.igem.org/Team%3AHong_Kong_HKUST/Model)

Parts

[Part Design](https://2019.igem.org/Team%3AHong_Kong_HKUST/Design)
[Basic Part](https://2019.igem.org/Team%3AHong_Kong_HKUST/Basic_Part)
[Part Collection](https://2019.igem.org/Team%3AHong_Kong_HKUST/Part_Collection)

[Results](https://2019.igem.org/Team%3AHong_Kong_HKUST/Results)

[Achievements](https://2019.igem.org/Team%3AHong_Kong_HKUST/Achievements)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AHong_Kong_HKUST/Human_Practices)
[Collaborations](https://2019.igem.org/Team%3AHong_Kong_HKUST/Collaborations)

[Our Team](https://2019.igem.org/Team%3AHong_Kong_HKUST/Team)

[Attributions](https://2019.igem.org/Team%3AHong_Kong_HKUST/Attributions)

 ☰

![](https://static.igem.org/mediawiki/2019/8/8d/T--Hong_Kong_HKUST--totop.svg)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Hong Kong HKUST/Experiments

Team:Hong Kong HKUST - 2019.igem.org

[![](https://static.igem.org/mediawiki/2019/9/99/T--Hong_Kong_HKUST--biscuitlogo.png)](https://2019.igem.org/Team%3AHong_Kong_HKUST)

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Project

[Description](https://2019.igem.org/Team%3AHong_Kong_HKUST/Description)
[Experiments](https://2019.igem.org/Team%3AHong_Kong_HKUST/Experiments)
[Application](https://2019.igem.org/Team%3AHong_Kong_HKUST/Application)
[Safety](https://2019.igem.org/Team%3AHong_Kong_HKUST/Safety)
[Logbook](https://2019.igem.org/Team%3AHong_Kong_HKUST/Logbook)

[Modelling](https://2019.igem.org/Team%3AHong_Kong_HKUST/Model)

Parts

[Part Design](https://2019.igem.org/Team%3AHong_Kong_HKUST/Design)
[Basic Part](https://2019.igem.org/Team%3AHong_Kong_HKUST/Basic_Part)
[Part Collection](https://2019.igem.org/Team%3AHong_Kong_HKUST/Part_Collection)

[Results](https://2019.igem.org/Team%3AHong_Kong_HKUST/Results)

[Achievements](https://2019.igem.org/Team%3AHong_Kong_HKUST/Achievements)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AHong_Kong_HKUST/Human_Practices)
[Collaborations](https://2019.igem.org/Team%3AHong_Kong_HKUST/Collaborations)

[Our Team](https://2019.igem.org/Team%3AHong_Kong_HKUST/Team)

[Attributions](https://2019.igem.org/Team%3AHong_Kong_HKUST/Attributions)

 ☰

![](https://static.igem.org/mediawiki/2019/8/8d/T--Hong_Kong_HKUST--totop.svg)

![experiments](https://static.igem.org/mediawiki/2019/5/5e/T--Hong_Kong_HKUST--experiments.svg)

### 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](https://static.igem.org/mediawiki/2019/3/39/T--Hong_Kong_HKUST--protocol.pdf)

# 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](https://static.igem.org/mediawiki/2019/a/a9/T--Hong_Kong_HKUST--RNA_protocols.pdf)

#### 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 |

![](https://static.igem.org/mediawiki/2019/3/3c/T--Hong_Kong_HKUST--RNAoverview.png)

Figure 1. Flow of RNA interaction assay

### Step 1 - PCR synthesis of IVT templates

The 2 RNA’s transcription templates, designed according to [**NEB IVT kit manual**](https://international.neb.com/protocols/2015/11/24/sgrna-synthesis-using-the-hiscribe-quick-t7-high-yield-rna-synthesis-kit-neb-e2050), were created by using PCR to insert a T7 promoter in front of the transcription template using an overhang forward primer. (Figure 2)

![](https://static.igem.org/mediawiki/2019/3/3e/T--Hong_Kong_HKUST--IVT_PCR_Primer.png)
Figure 2. RNA’s transcription templates PCR illustration

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:

![](https://static.igem.org/mediawiki/2019/9/98/T--Hong_Kong_HKUST--Expfig3.png)
Figure 3. Verification of RNA’s transcription templates

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](https://international.neb.com/products/m0303-dnase-i-rnase-free#Product%20Information)**, 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:

![](https://static.igem.org/mediawiki/2019/d/de/T--Hong_Kong_HKUST--Expfig4.png)
Figure 4. RNA transcription check

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](https://static.igem.org/mediawiki/2019/a/a9/T--Hong_Kong_HKUST--RNA_protocols.pdf)**)

It is known that salt content is crucial to RNA duplex formation [[1]](#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:

![](https://static.igem.org/mediawiki/2019/5/51/T--Hong_Kong_HKUST--RNA_hybridized2.png)
Figure 5. RNA hybridization assay

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) |

\*slow cool is done by cooling the RNA sample using thermo cycler at 5°C per 10 sec until reaching incubation temperature

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]](#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](https://static.igem.org/mediawiki/2019/e/e0/T--Hong_Kong_HKUST--SDSPAGE.docx)

![](https://static.igem.org/mediawiki/2019/e/e3/T--Hong_Kong_HKUST--Expfig6.png)
Figure 6. PCR verification of dCas9 transformed cells

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).

![](https://static.igem.org/mediawiki/2019/0/07/T--Hong_Kong_HKUST--Expfig7.png)
 Figure. 7 BSA standard curve and calculated protein concentration of lysis

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).

![](https://static.igem.org/mediawiki/2019/f/fd/T--Hong_Kong_HKUST--Expfig8.png)
Figure 8. dCas9 expression check with SDS-PAGE and Coomassie Blue staining

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.

![](https://static.igem.org/mediawiki/2019/4/42/T--Hong_Kong_HKUST--Expfig9.png)
Figure 9. dCas9 expression check with SDS-PAGE and Western Blot

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

![](https://static.igem.org/mediawiki/parts/8/84/T--Hong_Kong_HKUST--dCas9_suppresion.png)

#### 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)](https://www.biorxiv.org/content/10.1101/290155v1.full)** [[3]](#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.

![](https://static.igem.org/mediawiki/parts/5/54/T--Hong_Kong_HKUST--asCharacteriazation1.png)

| 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]](#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](https://static.igem.org/mediawiki/2019/e/e7/T--Hong_Kong_HKUST--old_part_protocol.pdf)

#### 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.

![](https://static.igem.org/mediawiki/2019/e/e9/T--Hong_Kong_HKUST--old1.png)

![](https://static.igem.org/mediawiki/2019/a/a9/T--Hong_Kong_HKUST--old2.png)

![](https://static.igem.org/mediawiki/2019/e/e3/T--Hong_Kong_HKUST--old3.png)

Our numerical results are tabulated as follows:

![](https://static.igem.org/mediawiki/2019/e/ee/T--Hong_Kong_HKUST--old4.png)

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.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Hong Kong HKUST/Results

Team:Hong Kong HKUST - 2019.igem.org

[![](https://static.igem.org/mediawiki/2019/9/99/T--Hong_Kong_HKUST--biscuitlogo.png)](https://2019.igem.org/Team%3AHong_Kong_HKUST)

×

Project

[Description](https://2019.igem.org/Team%3AHong_Kong_HKUST/Description)
[Experiments](https://2019.igem.org/Team%3AHong_Kong_HKUST/Experiments)
[Application](https://2019.igem.org/Team%3AHong_Kong_HKUST/Application)
[Safety](https://2019.igem.org/Team%3AHong_Kong_HKUST/Safety)
[Logbook](https://2019.igem.org/Team%3AHong_Kong_HKUST/Logbook)

[Modelling](https://2019.igem.org/Team%3AHong_Kong_HKUST/Model)

Parts

[Part Design](https://2019.igem.org/Team%3AHong_Kong_HKUST/Design)
[Basic Part](https://2019.igem.org/Team%3AHong_Kong_HKUST/Basic_Part)
[Part Collection](https://2019.igem.org/Team%3AHong_Kong_HKUST/Part_Collection)

[Results](https://2019.igem.org/Team%3AHong_Kong_HKUST/Results)

[Achievements](https://2019.igem.org/Team%3AHong_Kong_HKUST/Achievements)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AHong_Kong_HKUST/Human_Practices)
[Collaborations](https://2019.igem.org/Team%3AHong_Kong_HKUST/Collaborations)

[Our Team](https://2019.igem.org/Team%3AHong_Kong_HKUST/Team)

[Attributions](https://2019.igem.org/Team%3AHong_Kong_HKUST/Attributions)

 ☰

![](https://static.igem.org/mediawiki/2019/8/8d/T--Hong_Kong_HKUST--totop.svg)

![](https://static.igem.org/mediawiki/2019/c/c1/T--Hong_Kong_HKUST--results.svg)

## Introducing a foundation advancement into iGEM

Combined CRISPRi and antisense RNA toggle switch is a new biological switch system and is novel to the iGEM community. It has the potential to replace switches that rely on repressor protein - operon pairs. To bring the switch to life we have:

* Introduced new basic parts into the iGEM part registry

+ Custom-designed sgRNA for this switch: BBa\_K3017001, BBaK3017002
+ Custom-designed asRNA for this switch: BBa\_K3017003, BBaK3017004

* Created a model circuit using the above parts, on target proteins GFP and RFP: BBa\_3017070

[Here's our part collection!](https://2019.igem.org/Team%3AHong_Kong_HKUST/Part_Collection)

## Demonstration of the switch with a mathematical model

Our switch is brought to life by the model we built. It is a descriptive model that characterizes the expression of the output proteins of our system, to evaluate the switch’s effectiveness and the elasticity of variables. Our model shows that the system will take around 4 hours to reach that state, which is shorter than the Collins switch.
[Check out our model here!](https://2019.igem.org/Team%3AHong_Kong_HKUST/Model)

## Characterization of RNAs

We characterised our new composite parts BBa\_K3017061 and BBa\_K3017060 for their transcription and their interaction as a pair.

* T7-BBa\_K3017061 and T7-BBaK3017060 were synthesised by PCR
* T7-BBa\_K3017061 and T7-BBaK3017060 were transcribed *in vitro*
* Transcripts of T7-BBa\_K3017061 and T7-BBaK3017060 were hybridized. Results show that the custom-designed sgRNA and asRNA pair hybridize as expected. We expect even higher hybridization rate *in vivo*.

[Click to see the details of our experiment!](https://2019.igem.org/Team%3AHong_Kong_HKUST/Experiments#charac)

## Characterization of dCas9

We obtained our *Sp.*dCas9 gene, new to iGEM, from a professor in our university. Since the plasmid was not compatible with standard assembly, we had to make extra efforts to achieve the assembly of our circuit:

* SalI cut site, 6XHis tag and terminator were added to the dCas9 gene by PCR
* Transformed plasmid was verified by colony PCR and restriction digestion check
* Expression of dCas9 was verified by SDS-PAGE, Coomassie Blue staining and Western Blot

[Here is how we got dCas9!](https://2019.igem.org/Team%3AHong_Kong_HKUST/Experiments#HYM)

## Characterization of Old Part BBa\_R0010

LacI-regulated promoter BBa\_R0010 is used in our model circuit. The relative expression levels of GFP under BBa\_R0010 at different pH values were implied. This characterization helps us assess how different environmental conditions affect promoter activity. This, we believe, would aid in future application development:

* A graph of fluorescence/OD against pH (pH 5-9) was plotted from the plate reader measurements
* We found out:

+ p*Lac* on its own shows leak expression while that with added glucose shows repressed activity
+ The overall trend of GFP expression decreases with an increase in pH

[This is how we characterized BBa\_R0010!](https://2019.igem.org/Team%3AHong_Kong_HKUST/Experiments#oldpart)

## Characterization of competition between constitutive promoters

Professor Fei SUN’s postdoctoral fellows pointed out that the three constitutive promoters in our proposed circuit may display competitive behaviour among each other, thus leading to reduced protein expression by our circuit. We conducted an experiment to assess the level of interference between different numbers of promoters:

* Plasmids containing 1, 2 and 3 constitutive promoter-RBS-repeats (BBa\_J23104-B0034) are constructed to control a *mrfp* gene BBa\_E1010
* A plot of Fluorescence/OD shows no notable correlation between the number of promoters present in the plasmid and the level of fluorescence protein expression

[Click to see our promoter competition assay!](https://2019.igem.org/Team%3AHong_Kong_HKUST/Human_Practices#promoter)

//After iGEM//

# Future Updates

Unfortunately, we did not manage to test out our full circuit before the wiki-freeze deadline :(
However, some of our team members are planning to continue doing experiments to confirm that our Biscuit does work! We'll update this wiki once we've progressed.