iGEM 2019 — Team GENAS_China

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# Team:GENAS China/Notebook

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* Judging ▾
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  + [Awards](/Team%3AGENAS_China/Awards)

In the notebook below, we give the following abbreviation:

### 1.Template Plasmid G001: pSSTG1 G002: pSSTG2 pRGm1/m2/m3: pRG with different promotors 2. Report Plasmid Naming format: G001/G002-IC35/IF55/IS37/E21/T25/G22 G001/G002: the template plasmid G001, G002 IC35/IF55/IS37/E21/T25/G22: the corresponding recombinase recognition sites and the terminators within IC35: phiC31-ECK120034435 G22: TG1-L3S3P22 IF55: INT5-ECK120010855 IS37: INT7-ECK120033737 IE21: INT8-ECK120030221 T25: INT10-ECK120020525 3. Testing Strain Naming format: G1/G2+ IC35/IF55/IS37/E21/T25/G22+A1/A2/A4 G1/G2: the template plasmid G001, G002 IC35/IF55/IS37/E21/T25/G22: same as the naming in report plasmid A1/A2/A4: RBSs with different translation strength 4. Chromoprotein expression sequence R: Red by expression of eforRed Y: Yellow by expression of amilGFP B: Blue by expression of amilCP 5. Flipping System pRG-Recombinase 1-Recombinase 2v A flipping system with 3 different states by using 2 different recombinases Weekly ProgressWeek One 14/07/2019-20/07/2019 1. Set up the project and confirm the project topic. Week Two 21/07/2019-27/07/2019 1. Design six [attB-Terminator-attP] sequences: phiC31-ECK120034435 TG1-L3S3P22 INT5-ECK120010855 INT7-ECK120033737 INT8-ECK120030221 INT10-ECK120020525 Send the sequences to synthesis. 2. Design plasmids pSSTG1(G001), pSSTG2(G002) as testing platform. Design the construction plan and primers.

![](https://static.igem.org/mediawiki/2019/2/29/T--GENAS_China--note1.jpg)

Figure 1: the structure of plasmid G001

![](https://static.igem.org/mediawiki/2019/a/a5/T--GENAS_China--note2.jpg)

Figure 2: the structure of plasmid G002
3. Construct the induced-expressive plasmid (G003) of recombinase phiC31 on the basis of plasmid pRG.

![](https://static.igem.org/mediawiki/2019/5/5b/T--GENAS_China--note3.jpg)

### Week Three

28/07/2019-03/08/2019
1. Do the human practice in Shenzhen Children’s Hospital
2. Construct the induced-expressive plasmids (G004-G008) of recombinases INT5, INT7, INT8, INT10, TG1 on the basis of plasmid pRG.
3. Mutate the sequence of -10 region in the pTAC promotor of the plasmid pRG, and construct the induced-expression devices pRGm1, pRGm2, pRGm3 which have different dynamic ranges.
4. Construct plasmids G009 and G010 on the basis of G001 and G002, respectively.

### Week Four

04/08/2019-10/08/2019
1. Construct the report plasmid (G001-IC35/E21/T25/G22) on the basis of plasmid G001.
![](https://static.igem.org/mediawiki/2019/c/c9/T--GENAS_China--note4.jpg)

2. Construct the report plasmid (G001-IC35/E21/T25/G22) on the basis of plasmid G002.

![](https://static.igem.org/mediawiki/2019/a/aa/T--GENAS_China--note5.jpg)

3. Culture the testing strains G1IC35A1, G2IC35A1, G1IT25A1, G2IT25A1, G1IG22A1, G2IG22A1, G1IE21A1, G2IE21A1.
4. Assemble sfGFP to pRGm1, pRGm2, pRGm3.

### Week Five

11/08/2019-17/08/2019
1. Apply qualitative test to strains G2IC35A1, G2IT25A1, G2IG22A1, G2IE21A1 and determine the validity of the recombinase expressing system as well as the state effect the reversal of terminator has on the downstream gene.
2. By flow cytometry, quantitatively test the relay devices constructed by recombinase phiC31 and INT10 (G2IC35A1, G2IT25A1). Prove the feasibility of the inducing system and confirm the dynamic ranges of the terminator switches.

### Week Six

18/08/2019-24/08/2019
Prepare and take part in CCiC (Conference of China iGEMer Community).

### Week Seven

25/08/2019-31/08/2019
1. Use steady-state method to test the biological relays G1IC35A1, G1IT25A1, G1IG22A1, G1IE21A1.
2. Standardize the intensity of the systems’ input signal by IPTG-gradient inducing pTAC-sfGFP through the steady-state method.
3. Apply simplified test on the biological relays G2IC35A1, G2IT25A1, G2IG22A1, G2IE21A1.
4. Construct the report plasmid (G001-IF55/IS37) on the basis of plasmid G002.

### Week Eight

01/09/2019-07/09/2019
1. Culture the testing strains G2IF55A1, G2IS37A1.
2. Qualitatively test G2IF55A1, G2IS37A1.
3. Assemble eforRed to G002-IE21, construct the plasmid G2IE21-R1
Assemble amilGFP to G002-IT25, construct the plasmid G2IE21-Y1
Assemble amilCP to G002-IC35, construct the plasmid G2IE21-B1

![](https://static.igem.org/mediawiki/2019/e/e4/T--GENAS_China--note6.jpg)

### Week Nine

08/09/2019-14/09/2019
1. Find the insufficiency of the chromoprotein expression level in plasmids G2IE21-R1，G2IC35-B1. Optimize the RBS of the plasmids.
2. Construct the induced-expression system which combines recombinase phiC31, INT8, INT10, TG1 with RBS component B0032, B0034, on the basis of pRG.

### Week Ten

15/09/2019-21/09/2019
1. Culture the testing strains G2IC35A2/A4, G2IT25A2/A4, G2IE21A2/A4, G2IG22A2/A4, and apply qualitative test on them.
2. Do human practice in Huaqiang North electronic market
3. Reconstruct the testing strains G2IC35A1, G2IT25A1, G2IE21A1, G2IG22A1

### Week Eleven

22/09/2019-28/09/2019
1. Repeat the simplified test for G2IC35A1, G2IT25A1, G2IG22A1, G2IE21A1
2. Use steady-state method to test the gradient-induced curve for pRGm1-sfGFP、pRGm2-sfGFP、pRGm3-sfGFP
3. Do public engagement in Shenzhen Middle School
4. Categorize the part

### Week Twelve

29/09/2019-05/10/2019
1. Build up the color report system G010-BYR, G010-RYB on the basis of G010
2. Build up the flipping system pRG-IC1-T1, pRG-T1-IC2, pRG-T1-E1 on the basis of pRG

![](https://static.igem.org/mediawiki/2019/0/0a/T--GENAS_China--note7.jpg)

3. Change a weak promotor for INT8
4. Start to make Wiki

### Week Thirteen

06/10/2019-12/10/2019
1. Repeat the quantitative inducing experiment for G2IC35A1, G2IT25A1, G2IG22A1
2. Test and debug two ADC (analog-digit converter) system
3. Perfect the computer program

### Week Fourteen

13/10/2019-19/10/2019
1. Do public engagement in Shenzhen Media Group
2. Complete the judging form
3. Finish Wiki

---

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# Team:GENAS China/Experiments

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* Judging ▾
  + [Judging Form](https://igem.org/2019_Judging_Form?team=GENAS_China)
  + [Awards](/Team%3AGENAS_China/Awards)

# Experiments

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# Team:GENAS China/Results

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  + [Part Collection](/Team%3AGENAS_China/Part_Collection)
* Human Practices ▾
  + [Overview](/Team%3AGENAS_China/HP_Overview)
  + [Integrated Human Practices](/Team%3AGENAS_China/Human_Practices)
  + [Public Engagement](/Team%3AGENAS_China/Public_Engagement)
* Judging ▾
  + [Judging Form](https://igem.org/2019_Judging_Form?team=GENAS_China)
  + [Awards](/Team%3AGENAS_China/Awards)

## Overview on result

This page demonstrates the detailed result of the following experimental
process.

* Design of the recognition sites
* Testing and filtering the alternative recombinase
* Response curve for the chosen recombinase
* Solution for cytotoxicity
* Design, construction and debug of the three-state ADC

## Design of the recognition sites

### 1. The feasibility of normal open (NO) contact

If we compare our genetic circuit with the electrical circuit, the normal
open (NO) in our project is that there is no expression in the normal state
and the expression will be activate only when there is recombinase binding
on the sites and flips (or cuts) the terminator.

The feasibility of genetic switches based on att-site and unidirectional
terminators has been demonstrated in a study published by the Tim Lu’s team
in 2013 (see reference). Besides, 2017 iGEM team Peking and Fudan also
develop a series of recombinase switches on the same principle.

We replicate the recombinase switch design of 2017 Peking, which is based
on recombinase phiC31 with att recognition sites and the unidirectional
terminator ECK120034435. We test the expression difference between with and
without recombinase. And the result meets our expectation, shown below

![](https://static.igem.org/mediawiki/2019/3/30/T--GENAS_China--res1.jpg)

Figure 1: the upper shows the mechanism of the NO contact, and the lower
graph shows the feasibility of the NO contact.

### 2. The comparison between two design of normal close (NC) contact

The normal close (NC) circuit in our project is opposite to the NO circuit,
that there will be expression in the normal state and the expression will
disappear recombinases bind to the recognition sites. Based on the
mechanism of the recombinase, gene translation unit should be placed
between two att recognition sites to realize the normally-close control of
this gene. To make inserting gene easier, we design BsaI enzyme cutting
site between att and the terminator, on the cis-element of our recombinase
switches.

At this time, we consider two different design for the recognition sites:
placing att sites equidirectionally or placing att sites oppositely, which
will cause flipping or cutting respectively. We carry out experiment to
test these two designs, the result is shown below.

![](https://static.igem.org/mediawiki/2019/e/e9/T--GENAS_China--res2.jpg)

Figure 2: the mechanism and the result of flipping

![](https://static.igem.org/mediawiki/2019/e/e6/T--GENAS_China--res3.jpg)

Figure 3: the mechanism and the result of cutting

Compare with the cutting design, flipping design has the following
advantages and disadvantages:

**Advantages:**

can proceed the reverse process by using RDF

**Disadvantages:**

the closed (flipped) gene still has the probability of expression

The flipped fragment may have a strong attenuating effect on the expression
of downstream gene

After comprehensive analysis, we adopt the design of equidirectionally
placing att recoginition sites in most of our following experiment.

## Testing and filtering the alternative recombinase

Given the use of more than one recombinase in our system, we increase the
diversity of our parts and construct six recombinase-terminator-based
switches. We co-transform the G002-based testing plasmids with their
corresponding recombinase expression plasmids. After adding IPTG for
inducer and culturing in EP tubes for 24 hours, we centrifuge the culturing
tubes and make the cells fluorescent under blue light to qualitatively,
preliminarily screen our recombinase switches. The results are shown below.

![](https://static.igem.org/mediawiki/2019/8/87/T--GENAS_China--exp9.png)

Figure 4: the result of preliminary screening of recombinase system. IBR is
the name of our switches (integrase-based relay), followed by the composite
parts (attB-TE-attP), integrase – indicates cells without inducing while
integrase + means with inducing.

By using flow cytometry, we also screen the recombinase systems in a
quantitative way, shown below, whose result is consistent with the above
qualitative screening.

The switching performance of normally open contact is quantitatively
described by fluorescence attenuation rate of GFP reporter gene in two
states. As a reference, we used a plasmid containing only one short
unintentional sequence between the promoter and RiboJ as a positive
control. The average intensity of bacterial fluorescence was quantified by
flow cytometry, and the final value was calculated as follows:

![](https://static.igem.org/mediawiki/2019/8/8b/T--GENAS_China--res5.jpg)

Figure 5: the result of quantitative screening by using flow cytometry

The experimental results of orthogonality are as follows, and the result
reveals a fine orthogonality:

![](https://static.igem.org/mediawiki/2019/1/1d/T--GENAS_China--res6.jpg)

Figure 6: The results on the diagonal line, which are yielded from the
valid pairs of recombinase and its binding sites, indicate shorter DNA
fragments than others in the same row. This proves the fine orthogonality.

## Response curve for the chosen recombinase

So far, we have only solved the switching and conduction problems of relay
contacts. But as a replay element, it should have a character of changing
its ON/OFF state once it reaches rated voltage. We hope our biological
relay can have the similar character, which makes it different from the
transcription-factor-based, traditional promoter genetic switch.

In the existing applications of recombinase, there is only a simple
response to high and low input levels, and the threshold of response is not
demarcated. In this study, we mainly explore three issues: whether the
recombinase system has a narrow enough hypersensitive response interval to
realize state leap (rather than slow change); Determine the factors
affecting the hypersensitive response range; Describe the range of
different modules quantitatively (that is, parameterized characterization).

We establish a mathematical model based on the thermodynamic equilibrium
state hypothesis (see model
[https://2019.igem.org/Team:GENAS\_China/Model](https://2019.igem.org/Team%3AGENAS_China/Model)
), shown below.

![](https://static.igem.org/mediawiki/2019/2/2d/T--GENAS_China--res7.jpg)

According to the framework of previous studies (see reference), the
intensity of Input was used to characterize the concentration of
recombinase in cell.

In the following simulation image, the Hill’s coefficient is set to 2
according to the hypothesis of previous studies, and the range of the x
axis is determined according to the measurement range of the input strength
of the Ptac promoter. The shape of the curve shows that the range of
hypersensitivity response is very narrow, which is helpful to realize the
response to the slight changes in the input signal.

Different k values will cause the change of the position of the
hypersensitive response range in the transfer function of module. The
following figures shows how the change in k value affect the transfer
function.

![](https://static.igem.org/mediawiki/2019/6/66/T--GENAS_China--res8.jpg)
![](https://static.igem.org/mediawiki/2019/8/8a/T--GENAS_China--res9.jpg)

Figure 7: different curve under different value of Kp and Kb

In order to make the measurement conditions as close as possible to the
theoretical hypothesis, we designed the measurement method 1 based on the
steady-state measurement method (that is, recombinase induction first,
followed by reporting system induction) and simplified method 2. We use
phiC31 recombinase-based relay to compare the two methods and
parameterizedly characterize the phiC31 relay system.

![](https://static.igem.org/mediawiki/2019/7/74/T--GENAS_China--res10.jpg)
![](https://static.igem.org/mediawiki/2019/4/46/T--GENAS_China--res11.jpg)

Figure 8:
the fitting response curve of phiC31 RBBR component.

The orange part are the parameters that need to be fitted.

Although the fitting results of the data obtained by these two measurement
methods are not identical, their transfer functions are very similar. In
order to simplify the experimental process, we will adopt the simplified
method 2 to conduct the measurement.

Using measurement method 2, we parameterizedly characterize the other two
RBBR elements, the parameters are shown below.

![](https://static.igem.org/mediawiki/2019/6/6f/T--GENAS_China--res12.jpg)
![](https://static.igem.org/mediawiki/2019/2/24/T--GENAS_China--res13.jpg)

Figure 9: the fitting response curve of phiC31, TG1 and INT10 RBBR
component.

## Solution for cytotoxicity

During the process of experiment, we find that the overexpression of some
recombinases will limit the growth of cells. The relationship between gene
expression level (represent in input strength below) and the cytotoxicity
(represent in OD600 below; the lower OD600, the higher cytotoxicity) is
shown in the graph below.

![](https://static.igem.org/mediawiki/2019/4/47/T--GENAS_China--exp13.png)

Figure 10: the relationship between OD600 and the input level.

In the figure, INT8 (red) and TG1 (green) have distinct cytotoxicity since
the OD600 value drop greatly when the input level increase.

To solve the problem of cytotoxicity, we try to lower the gene expression
strength by either changing a weak promoter or finetuning RBS to a weak
one. We lower the expression strength in INT8 as an example of changing a
weak promoter.

For INT8, when the input is above 30000, the cell growth will be
significantly inhibited, and other experiments have proved that the Int8
can lead to sufficient recombination even if the input is the lowest value
of this promoter. Suppose our task is to design an Int8 induction system
that is not toxic at the highest inducer concentration. To this end, we
mutated the original promoter and obtained the following promoter:

![](https://static.igem.org/mediawiki/2019/6/61/T--GENAS_China--res15.jpg)

Figure 11: different promoter with different relationship between
concentration of IPTG and expression of sfGFP. Ptac-M1/M2/M3 are the
mutated promoter, which are relatively weaker than the original one.

The combination of promoter ptac-m2 and K3254011 which has the highest
output below 20000 is selected, and the results showed that the toxicity
was successfully controlled, as shown in the figure below (in blue line),
the OD600 value still remains high when the concentration increase
(Although the leakage expression is still high, it has been fully
recombinated without inducers.)

![](https://static.igem.org/mediawiki/2019/b/ba/T--GENAS_China--res16.jpg)

Figure 12: different result of cytotoxicity before and after the change of
promoter.

Therefore, when we evaluate a bio-relay element, we need to evaluate the
performance of the whole system (Figure 13)

![](https://static.igem.org/mediawiki/2019/7/70/T--GENAS_China--res17.jpg)

Figure 13: comprehensive prediction and evaluation of a system’s
performance.

## Design, construction and debug of the three-state ADC

Based on the biological relay we developed in the project, we built a
genetic analog-digital convertor, which can convert the analog input to the
digital output. Each recombinase correspondingly controls the on-off of a
chromoprotein. Theoretically, the convertor can be infinitely extensive by
adding new relays to the system. The performance of the ADC predicted by
the model is presented in figure 14 and 15. The amilCP is the first
fluorescent protein that is activated by the downstream promoter, and hence
it’s constantly expressed in the beginning. amilGFP is then activated
during a short period till mCherry start to express.

![](https://static.igem.org/mediawiki/2019/e/ed/T--GENAS_China--res18.jpg)

Figure 14: a genetic circuit demonstration of relay-based ADC

However, the experimental result in figure 15 (upper) doesn’t fit with our
simulation. amilCP’s blue color did vanish in expectation but yellow and
red color didn’t appear as we predicted. The disappearance of mCherry might
be due to the low transcription efficiency caused by redundancy of upstream
sequence. But for the disappearance of amilCP, the PCR result showed that
at the minimum inducer concentration, amilCP have already been
recombinated, indicating that the Int10 might have strong leaked expression
in the system. This might due to the upstream phiC31 sequence, an original
sequence adapted from phage, which may be multifunctional because of the
limited length of DNA in a phage. Hence, there might be one or more
potential promoter in phiC31 sequence which may cause the leaked expression
of amilGFP

![](https://static.igem.org/mediawiki/2019/d/df/T--GENAS_China--res19.jpg)

Figure 15: the comparison between stimulation of the performance of ADC
(lower) and the experimental result (upper).

**Reference**

[1] Lou, C., et al., Ribozyme-based insulator parts buffer synthetic
circuits from genetic context. Nature Biotechnology, 2012. 30(11): p.
1137-42.

[2] Zhang, H.M., et al., Measurements of Gene Expression at Steady State
Improve the Predictability of Part Assembly. ACS Synthetic Biology, 2016.
5(3): p. 269-273.

[3] Zong, Y., et al., Insulated transcriptional elements enable precise
design of genetic circuits. Nat Commun, 2017. 8(1): p. 52.

---

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