Difference between revisions of "Team:XJTU-China/Engineering"
| Line 111: | Line 111: | ||
</div> | </div> | ||
</div> | </div> | ||
| − | <!-- 2. in-silico engineering --> | + | <!-- 2. <i>in-silico</i> engineering --> |
<div class="col-12"> | <div class="col-12"> | ||
<a class="anchor" id="2"></a> | <a class="anchor" id="2"></a> | ||
| − | <h2 class="mt-5 pl-3">2. <i> | + | <h2 class="mt-5 pl-3">2. <i><i>in-silico</i></i> Engineering</h2> |
<a class="anchor" id="2.1"></a> | <a class="anchor" id="2.1"></a> | ||
<h3 class="pl-5">2.1 Design, Build and Test</h3> | <h3 class="pl-5">2.1 Design, Build and Test</h3> | ||
| − | <p class="mt-3">Before our experiment, modeling was performed for in-silico prediction | + | <div class="row"> |
| − | + | <div class="col-md-5 col-12"> | |
| − | + | <p class="mt-3">Before our experiment, modeling was performed for <i><i>in-silico</i></i> | |
| − | + | prediction | |
| − | + | of behaviours of the toggle switch circuit. We constructed a equation set | |
| − | + | based on | |
| − | + | transcription and expression to describe the changes in the relative | |
| − | + | expression | |
| − | + | levels of GFP and RFP under different inducing conditions(IPTG induction or | |
| − | + | 42℃ | |
| − | + | heat). Equations used are listed in Fig. 2.1</p> | |
| − | + | <p>The <i>in silico</i> result of the expression level of <i>sfGFP</i> and <i>mRFP</i> under | |
| − | + | different leaky expression factor α<span class="sub">0</span> as shown in Fig2.2， which indicates | |
| + | that the behavior of toggle switch is largely affected by the leaky | ||
| + | expression of two promoters. A biostability can be achieved under low α<span class="sub">0</span>, | ||
| + | and toggle switch will fail in case of high α<span class="sub">0</span>. </p> | ||
| + | <div class="imgWrapper centerize"> | ||
| + | <img src="https://static.igem.org/mediawiki/2021/e/e9/T--XJTU-China--result_2.png" | ||
| + | alt="the model of toggle switch" width="90%"> | ||
| + | <span class="description"><strong>Fig. 2.2 Toggle Switch Modelling | ||
| + | Result.</strong> | ||
| + | IPTG is | ||
| + | introduced at 1000 min | ||
| + | and the induction condition is switched to 42℃ at 2000 min.</span> | ||
| + | </div> | ||
| + | |||
| + | </div> | ||
| + | <div class="col-md-7 col-12"> | ||
| + | <div class="imgWrapper centerize"> | ||
| + | <img src="https://static.igem.org/mediawiki/2021/2/24/T--XJTU-China--engineering-equations.png" | ||
| + | alt="the result of toggle switch modelling" width="90%"> | ||
| + | <span class="description"><strong>Fig. 2.1 Toggle Switch Modelling | ||
| + | Equations.</strong></span> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | </div> | ||
| + | |||
| + | |||
| + | |||
| + | |||
<a class="anchor" id="2.2"></a> | <a class="anchor" id="2.2"></a> | ||
<h3 class="mt-3 pl-5">2.2 Learn</h3> | <h3 class="mt-3 pl-5">2.2 Learn</h3> | ||
| − | <p class="mt-3">By analyzing the result of our in-silico test, we concluded that | + | <p class="mt-3">By analyzing the result of our <i>in-silico</i> test, we concluded that |
avoiding | avoiding | ||
leakage expression of repressors is the key to successfully construct the | leakage expression of repressors is the key to successfully construct the | ||
| Line 201: | Line 229: | ||
<a class="anchor" id="3.3.1"></a> | <a class="anchor" id="3.3.1"></a> | ||
<h4 class="pl-5">3.3.1 RT-qPCR quantifications of Toggle-Switch</h4> | <h4 class="pl-5">3.3.1 RT-qPCR quantifications of Toggle-Switch</h4> | ||
| − | <p>We first used RT-qPCR to detect the transcription of each gene under different | + | <p class="mt-3">We first used RT-qPCR to detect the transcription of each gene under |
| + | different | ||
induction conditions, so as to preliminarily judge the effect of promoter | induction conditions, so as to preliminarily judge the effect of promoter | ||
bistable | bistable | ||
| Line 264: | Line 293: | ||
<a class="anchor" id="3.3.2"></a> | <a class="anchor" id="3.3.2"></a> | ||
<h4 class="mt-3 pl-5">3.3.2 Characterization</h4> | <h4 class="mt-3 pl-5">3.3.2 Characterization</h4> | ||
| − | <div class="card card-dark ml-5 mt- | + | <div class="card card-dark ml-5 mt-5" style="width: 90%;"> |
<button class="btn btn-default" type="button" data-toggle="collapse" | <button class="btn btn-default" type="button" data-toggle="collapse" | ||
| − | data-target="#characterization-method" aria-expanded="false" aria-controls="part"> | + | data-target="#characterization-method" aria-expanded="false" |
| + | aria-controls="part"> | ||
Method | Method | ||
</button> | </button> | ||
| Line 272: | Line 302: | ||
<div class="card card-body card-dark"> | <div class="card card-body card-dark"> | ||
<p class="mt-3"> | <p class="mt-3"> | ||
| − | <b style="font-size: 1.2em;">Method:</b><br> Due to the expression of reporter genes in toggle | + | <b style="font-size: 1.2em;">Method:</b><br> Due to the expression of |
| + | reporter genes in toggle | ||
switch, GFP | switch, GFP | ||
and | and | ||
Revision as of 07:19, 20 October 2021
Engineering Success
Toggle Switch
1. General Design
This circuit is an updated version of the traditional toggle-switch developed by James J. Collins's group in 2000 (Gardner et al. Nature, 2000), which contains two feedbacks each controls the other, and can achieve bistability of protein expression in different inducing conditions.
Two promoter-repressor systems (lacUV5 promoter-LacI and lambda-CI857) with sfGFP and mRFP respectively, was constructed in our updated version, to monitor the two states of the circuit. With induction of IPTG, the downstream genes of lacUV5, that is, CI protein and mRFP will expressed, while those in the downstream of lambda promoter (LacI and sfGFP) will be repressed. Even without IPTG induction after several hours, the lack of LacI expression will result in the stability of red fluorescence. At temperatures above 42 degrees Celsius, gene expression will be flipped into another state, the stable expression of LacI and sfGFP, and the state will maintain even without heat. The GFP and RFP can be altered with other functional genes such as tyrptohan synthesitic genes to achieve the bistable expression and synthesis of tyrptohan.
The design of toggle-switch circuit is shown in Fig. 1.1.
Fig. 1.1 Design of Toggle-Switch
Circuit
2. in-silico Engineering
2.1 Design, Build and Test
Before our experiment, modeling was performed for in-silico prediction of behaviours of the toggle switch circuit. We constructed a equation set based on transcription and expression to describe the changes in the relative expression levels of GFP and RFP under different inducing conditions(IPTG induction or 42℃ heat). Equations used are listed in Fig. 2.1
The in silico result of the expression level of sfGFP and mRFP under different leaky expression factor α0 as shown in Fig2.2， which indicates that the behavior of toggle switch is largely affected by the leaky expression of two promoters. A biostability can be achieved under low α0, and toggle switch will fail in case of high α0.
Fig. 2.2 Toggle Switch Modelling
Result.
IPTG is
introduced at 1000 min
and the induction condition is switched to 42℃ at 2000 min.
Fig. 2.1 Toggle Switch Modelling
Equations.
2.2 Learn
By analyzing the result of our in-silico test, we concluded that avoiding leakage expression of repressors is the key to successfully construct the toggle-switch circuit. This would provide guidance for our following experiment design, including selecting the appropriate RBSs to build our circuit.
3. Experimental Engineering
3.1 Design
Generally, the toggle-switch circuit is constructed utilizing two sets of promoter-repressor system, namely, lacUV5 promoter-LacI and lambda promoter-cI857, in addition to sfGFP and mRFP as the reporter genes (see the Fig.1.1). Promoter lacUV5 initiates the transcription of the downstream genes cI857 and mRFP, leading to a combined result of red fluorescence and pλ inhibition. While the pλ, just the contrary, starts the expression of GFP and lacUV5 repressor LacI. Thus, if inducing the cells with IPTG, the inhibition on lacUV5 by LacI will be released, triggering the generation of red signal. And the treatment of higher incubation temperature, like 42℃, promotes the degradation of cI protein, inducing the biological function of pλ then causing a green signal.
3.2 Build
In our project, Golden Gate Assembly (GG) is applied to ligate all the genes and their backbones pET28a+. Thus, a series of DNA fragments with flanking sequences which are consistent type IIS restriction enzyme recognition sites and complementary restriction sites are generated using PCR amplification. Additionally, the 5’ overhang of primers may also extend the amplicons with some short but fundamental parts like Shine-Dalgarno sequences, promoters and terminators.
The figure of agarose gel electrophoresis of DNA fragments that build the toggle-switch circuit is shown below.
Fig. 3.1 Agarose gel electrophoresis of
toggle-switch circuit
These two fragments are consequently used for GG to construct the plasmid containing our toggle-switch circuit. After transformation to E.coli DH5alpha, plasmid extraction and a set of screening including colony PCR and double digestion, strains with expected plasmid are selected, indicating the toggle-switch circuit is built.
Fig. 3.2 Agarose gel electrophoresis of
cloned plasmid inserted with toggle-switch circuit
3.3 Test
3.3.1 RT-qPCR quantifications of Toggle-Switch
We first used RT-qPCR to detect the transcription of each gene under different induction conditions, so as to preliminarily judge the effect of promoter bistable expression.
Method:
Cultivation: Using LB liquid medium, 37℃, 200rpm. After
cultivating for 3h,
cells
are
cultivated under inducing conditions (1mM IPTG / 42℃) for 8h
respectively.
Total RNA extraction: Using RNAsimple Total RNA Kit,DP419 (TIANGEN
BIOTECH (BEIJING)
CO.,LTD.)
cDNA preparation: Using Evo M-MLV RT Mix (Vazyme Biotech Co.,Ltd);
template
concentration: 50ng RNA/ul; reaction condition: 37℃ 15min,
85℃
15sec.
qPCR: Using ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd).
Relative Normalized Expression data is calculated by using the equation
below,
Relative Expression = 2-[ΔCt(T)-ΔCt(C)]
where ΔCt(T) represents the difference between
Ct value of target gene and internal standard
gene in
treatment
group; ΔCt(C) represents the difference between
Ct value of target gene and internal standard
gene in negative
control group.
Results: The group induced by 1mM IPTG represents obliviously up-regulation of transcription of mRFP and cI857 while inhibited in sfGFP and lacI; under 42℃ relative expression of this genes has been reversed comparing induced by IPTG (Fig.3.3).
Fig. 3.3 Relative normalized expression
of
(a): mRFP and sfGFP (b): cI857 and lacI in toggle-switch circuit measured by
RT-qPCR
3.3.2 Characterization
Method:
Due to the expression of
reporter genes in toggle
switch, GFP
and
RFP
signals are detected by a microplate reader (SpectraMax i3). After
normalization,
that is, dividing the fluorescent signals by corresponding OD600, the intensity of
fluor-signal per cell is gained. And upon inducing with either IPTG or
42℃,
temporal
determination of all the OD600, GFP &RFP
fluorescent
signals provides us a set of
data that could describe the bistable-switch function of our
toggle-switch
circuit.
We used full spectrum scanning to determine the most appropriate
excitation and
emission wavelengths for the two fluorescent proteins. Wavelength used
are as
below:
sfGFP: Excitation 485nm; Emission 535nm
mRFP: Excitation 587nm; Emission 627nm
Cultivation: Using LB liquid medium, 37℃, 200rpm.
Induction: 1mM IPTG was used in 0-719 min culturing. Then the cells were
collected
by centrifugation and replaced with fresh medium without IPTG, and
cultured at
42℃
The obtained fluorescence intensity data was normalized by subtracting the intensity of the negative control (DH5alpha strain containing empty pET8a+ vector) and divided by the 600nm absorbance (representing the thallus concentration).
Result:
As in Fig.3.4, the relative fluorescence intensity of sfGFP and mRFP reversed
after
changing the induction conditions.
Fig. 3.4 Relative fluorescence
intensity
of sfGFP and mRFP in toggle-switch circuit under different inducing
conditions
3.4 Learn
Conclusion: We confirmed the function of toggle-switch circuit in RT-qPCR and fluorescence measurement results. We successfully achieved the bistable expression of sfGFP and mRFP under different induction conditions. The alternation of the two fluorescence intensities was observed after the change of conditions, reporting the intensities of respective promoters.
According to the above results,the feasibility has been proved that we can use this circuit to control the expression of aroG, trpBA and pykA genes, so as to control the cell into different states of “proliferation” and “production”.