iGEM 2020 — Team OUC-China

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:OUC-China/Notebook

[![](https://static.igem.org/mediawiki/2020/7/73/T--OUC-China--middle_button_on.png)](https://2020.igem.org/Team%3AOUC-China)

* PROJECT
  + [Description](https://2020.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2020.igem.org/Team%3AOUC-China/Design)
  + [Results](https://2020.igem.org/Team%3AOUC-China/Results)
  + [Contribution](https://2020.igem.org/Team%3AOUC-China/Contribution)
  + [Engineering](https://2020.igem.org/Team%3AOUC-China/Engineering)
  + [Implementation](https://2020.igem.org/Team%3AOUC-China/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3AOUC-China/Proof_Of_Concept)
* WET LAB
  + [Experiments](https://2020.igem.org/Team%3AOUC-China/Experiments)
  + [Safety](https://2020.igem.org/Team%3AOUC-China/Safety)
  + [Measurement](https://2020.igem.org/Team%3AOUC-China/Measurement)
* [MODEL](https://2020.igem.org/Team%3AOUC-China/Model)
  + [Overview](https://2020.igem.org/Team%3AOUC-China/Model#model_ov_po)
  + [3WJ](https://2020.igem.org/Team%3AOUC-China/Model#model_3wj)
  + [ODE](https://2020.igem.org/Team%3AOUC-China/Model#model_ode)
  + [Sequence Design](https://2020.igem.org/Team%3AOUC-China/Model#model_sd)
  + [Software](https://2020.igem.org/Team%3AOUC-China/Software)
* [PARTS](https://2020.igem.org/Team%3AOUC-China/Parts)
  + [Overview](https://2020.igem.org/Team%3AOUC-China/Parts)
  + [Improvement](https://2020.igem.org/Team%3AOUC-China/Parts#part_impro)
  + [Collection](https://2020.igem.org/Team%3AOUC-China/Part_Collection)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AOUC-China/Human_Practices)
  + [Human Practices](https://2020.igem.org/Team%3AOUC-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AOUC-China/Education)
  + [Comics](https://2020.igem.org/Team%3AOUC-China/Comics)
* [COLLABORATIONS](https://2020.igem.org/Team%3AOUC-China/Collaborations)
  + [Collaborations](https://2020.igem.org/Team%3AOUC-China/Collaborations)
  + [Meet Up](https://2020.igem.org/Team%3AOUC-China/Collaborations#col_mu)
  + [Team List](https://2020.igem.org/Team%3AOUC-China/Collaborations#col_tl)
  + [Partnership](https://2020.igem.org/Team%3AOUC-China/Partnership)
* [TEAM](https://2020.igem.org/Team%3AOUC-China/Team)
  + [Members](https://2020.igem.org/Team%3AOUC-China/Team)
  + [Attributions](https://2020.igem.org/Team%3AOUC-China/Attributions)

[![](https://static.igem.org/mediawiki/2020/0/0a/T--OUC-China--back_to_top.png)](#toptop)

# 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://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:OUC-China/Engineering

[![](https://static.igem.org/mediawiki/2020/7/73/T--OUC-China--middle_button_on.png)](https://2020.igem.org/Team%3AOUC-China)

* PROJECT
  + [Description](https://2020.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2020.igem.org/Team%3AOUC-China/Design)
  + [Results](https://2020.igem.org/Team%3AOUC-China/Results)
  + [Contribution](https://2020.igem.org/Team%3AOUC-China/Contribution)
  + [Engineering](https://2020.igem.org/Team%3AOUC-China/Engineering)
  + [Implementation](https://2020.igem.org/Team%3AOUC-China/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3AOUC-China/Proof_Of_Concept)
* WET LAB
  + [Experiments](https://2020.igem.org/Team%3AOUC-China/Experiments)
  + [Safety](https://2020.igem.org/Team%3AOUC-China/Safety)
  + [Measurement](https://2020.igem.org/Team%3AOUC-China/Measurement)
* [MODEL](https://2020.igem.org/Team%3AOUC-China/Model)
  + [Overview](https://2020.igem.org/Team%3AOUC-China/Model#model_ov_po)
  + [3WJ](https://2020.igem.org/Team%3AOUC-China/Model#model_3wj)
  + [ODE](https://2020.igem.org/Team%3AOUC-China/Model#model_ode)
  + [Sequence Design](https://2020.igem.org/Team%3AOUC-China/Model#model_sd)
  + [Software](https://2020.igem.org/Team%3AOUC-China/Software)
* [PARTS](https://2020.igem.org/Team%3AOUC-China/Parts)
  + [Overview](https://2020.igem.org/Team%3AOUC-China/Parts)
  + [Improvement](https://2020.igem.org/Team%3AOUC-China/Parts#part_impro)
  + [Collection](https://2020.igem.org/Team%3AOUC-China/Part_Collection)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AOUC-China/Human_Practices)
  + [Human Practices](https://2020.igem.org/Team%3AOUC-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AOUC-China/Education)
  + [Comics](https://2020.igem.org/Team%3AOUC-China/Comics)
* [COLLABORATIONS](https://2020.igem.org/Team%3AOUC-China/Collaborations)
  + [Collaborations](https://2020.igem.org/Team%3AOUC-China/Collaborations)
  + [Meet Up](https://2020.igem.org/Team%3AOUC-China/Collaborations#col_mu)
  + [Team List](https://2020.igem.org/Team%3AOUC-China/Collaborations#col_tl)
  + [Partnership](https://2020.igem.org/Team%3AOUC-China/Partnership)
* [TEAM](https://2020.igem.org/Team%3AOUC-China/Team)
  + [Members](https://2020.igem.org/Team%3AOUC-China/Team)
  + [Attributions](https://2020.igem.org/Team%3AOUC-China/Attributions)

[![](https://static.igem.org/mediawiki/2020/0/0a/T--OUC-China--back_to_top.png)](#toptop)

![](https://static.igem.org/mediawiki/2020/f/fb/T--OUC-China--engineering_banner.jpg)

# Engineering Design Cycle

Inspiration → Research → Imagine → Design → Test → Improve → Build → Test → Improve → Build → Simulate → Build → Test → Simulate → Design → Simulate → Future

## Inspiration:

Our school is located in Qingdao, a coastal city with a wet atmosphere. That’s not good news for computers, for the high air humidity can impact their service life. Our computer equipment often breaks down because of the high humidity. Apart from this, there are also other environmental factors that interrupt its normal function, like, temperature and dust, etc. What’s more, the thickness of silicon chips has long been a factor affecting computers’ performance. In the future, perhaps we need to seek other kinds of computers that can overcome these disadvantages, and a biocomputer is an option.

## Research：

The ultimate goal of combining molecular biology and engineering is to realize the biocomputer. Since the electronic computer is so well-developed according to our research, it seemed unlikely that the biocomputer element we research into will easily surpass the calculation speed of electronic computers.

Therefore, biocomputer should demonstrate excellence in another area, such as, high humidity tolerance, high biocompatibility and mass storage capacity. Biocomputer should have special functions which emphasis on biological characteristic and would be complementary with electronic computers eventually. The expertise we obtained has inspired us to consider realizing biological functions with our biocomputer designs. Finally, in the discussion with other iGEM teams in meet up, we settled that our logic gate designs can be applied to detecting viruses.

## Imagine：

Our project is not trying to solve a real world problem with practical applications right away, we built elements of a biological computer. Logic gates are the foundation of computing. To build a biocomputer, we need to start from the basic parts built by biological elements. We hope it will have scientific values and can serve as a comprementary role to electronic computers in the future.

## Design：

We chose toehold as the ON-switch and three-way Junction (3WJ) repressor as the OFF-switch. And we used it in the construction of logic gates and the design of some more complex circuits.

## Test：

We conducted a series of tests on promoters, the ON RNA switch and the OFF RNA switch. The results of these basic tests provided references for the construction of logic gates and the design of some more complex circuits.

## Improve：

We hope to improve the stability of the trigger from two aspects to reduce false negatives.

① Add the hairpin at the 5' end of the trigger.

② Knockout/knockdown of RNAase gene (using *E. coli* BL21 Star (DE3) as biological chassis).

## Build：

Based on toehold, 3WJ repressor and existing logic gates, we designed new logic operations (IMPLY gate and XOR gate).

## Test：

In the verification of the logic circuit, the inductors corresponding to the promoter before the switch structure were taken as the inputs, and the expression quantity of fluorescent protein was taken as the output, so as to test the existing logic operations and the logic operations designed by us (IMPLY gate and XOR gate).

## Improve：

We have developed a [SOFTWARE](https://2020.igem.org/Team%3AOUC-China/Software) that can calculate the common threshold. Threshold calculation can give the normalized parameters of fluorescence intensity and the calculated threshold, so that the designers can get the common threshold of different logic gates easily and combine different types of logic gates better.

## Build：

We used σ factors to implement cascades of different RNA logic gates. We also designed more complex multiple-input logic gates and swapping gates to show more possibilities of logic gates.

## Simulate：

We use universal thresholds, promoter data, basic logic gate data and σ factor data to build cascade loops, multiple-input gates and swapping gates.

## Build：

We construct the adder and the subtractor with our logic gates. The calculation can be done by adjusting the concentration of trigger RNA. In the future, they can be used in the more complicated calculations.

## Test：

Test the logic gates needed (XOR gate, AND gate and NIMPLY gate) to build half adder and subtracter.

## Simulate：

We simulate the operations of semi-adders and subtracters with available data.

## Design：

Our logic gates also can be used to detect various viruses. We design the logic operations according to the virus sequence. The use of them can reduce false negatives and false positive of virus tests and also makes it possible to detect multiple viruses simultaneously.

## Simulate：

We tested the matching degree of the logic operation and the virus sequence by modeling method. The logic operations can identify the sequence well and detect it.

## Future：

Under the influence of COVID-19, our experimental time was more limited and the control of virus usage became stricter. Therefore, we didn’t conduct experimental verification on the application of virus detection, but only demonstrated the application of virus detection with the logic gates in the biological computer through design and modeling simulation. We believe that these logic gates have great potential in resistance gene detection, virus detection, and disease detection.

# New Parts

We designed and built some new parts, and they worked as expected.

## [BBa\_K3328035](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3328035)

The XOR is inspired by the NIMPLY gate, consisting of a toehold switch and two triggers.

![](https://static.igem.org/mediawiki/2020/8/86/T--OUC-China--design_fig7.jpg)

Figure 1. Demonstration of XOR gate

The trigger’s core sequence is the same and at the triggers’ both ends there are the nucleotide-binding domains. When input one of these triggers, the switch can turn on. Moreover, when input these two triggers simultaneously, they can pair together and form a ring in the middle. As a result, the switch will still be in the OFF state.

![](https://static.igem.org/mediawiki/2020/1/14/T--OUC-China--part_XOR2.jpg)

Figure 2. Two-input toehold XOR gate

The left figure shows that when INPUT A and INPUT B are both 0 or 1, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other two groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/ml) is added, INPUT B=1 means that HSL (0.1 mg/ml) is added.

## [BBa\_K3328041](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3328041)

We combined the 3WJ switch and toehold switch to realize the IMPLY Boolean calculation.

![](https://static.igem.org/mediawiki/2020/f/f7/T--OUC-China--design_lunbo_imply.jpg)

Figure 3. Demonstration of IMPLY gate

When no trigger is expressed, this logic gate just likes a 3WJ switch. When trigger A expressed, the trigger will bind to the switch RNA. The binding allows for a branch migration process, exposing AUG and RBS for translation initiation. When trigger B expressed, the trigger will bind to 3WJ switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and the start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation. When both trigger A and B are expressed, the binding of trigger RNA to the toehold sequence allows the RNA polymerase to bind to the former RBS and break open 3WJ stable hairpin.

![](https://static.igem.org/mediawiki/2020/6/66/T--OUC-China--part_IMPLY2.jpg)

Figure 4. Two-input toehold and 3WJ repressor IMPLY gate

The left figure shows that when INPUT A=0, INPUT B=1, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/ml) is added, INPUT B=1 means that HSL (0.1 mg/ml) is added.

## [BBa\_K3328050](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3328050)

We improved the NIMPLY gate in the paper and built new parts.

![](https://static.igem.org/mediawiki/2020/8/87/T--OUC-China--design_lunbo_nimply.jpg)

Figure 5. Demonstration of NIMPLY gate

In the NIMPLY gate, a deactivating RNA (INPUT A) uses direct hybridization or strand displacement to abolish trigger RNA (INPUT B) activity. So when only the correct trigger RNA is expressed, the switch can be turned on.

![](https://static.igem.org/mediawiki/2020/1/1f/T--OUC-China--part_NIMPLY2.jpg)

Figure 6. Two-input 3WJ repressor NIMPLY gate

The left figure shows that when INPUT A=0, INPUT B=1, the fluorescence of GFP is high. And the fluorescence intensity of GFP was low in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/ml) is added, INPUT B=1 means that HSL (0.1 mg/ml) is added.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:OUC-China/Experiments

[![](https://static.igem.org/mediawiki/2020/7/73/T--OUC-China--middle_button_on.png)](https://2020.igem.org/Team%3AOUC-China)

* PROJECT
  + [Description](https://2020.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2020.igem.org/Team%3AOUC-China/Design)
  + [Results](https://2020.igem.org/Team%3AOUC-China/Results)
  + [Contribution](https://2020.igem.org/Team%3AOUC-China/Contribution)
  + [Engineering](https://2020.igem.org/Team%3AOUC-China/Engineering)
  + [Implementation](https://2020.igem.org/Team%3AOUC-China/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3AOUC-China/Proof_Of_Concept)
* WET LAB
  + [Experiments](https://2020.igem.org/Team%3AOUC-China/Experiments)
  + [Safety](https://2020.igem.org/Team%3AOUC-China/Safety)
  + [Measurement](https://2020.igem.org/Team%3AOUC-China/Measurement)
* [MODEL](https://2020.igem.org/Team%3AOUC-China/Model)
  + [Overview](https://2020.igem.org/Team%3AOUC-China/Model#model_ov_po)
  + [3WJ](https://2020.igem.org/Team%3AOUC-China/Model#model_3wj)
  + [ODE](https://2020.igem.org/Team%3AOUC-China/Model#model_ode)
  + [Sequence Design](https://2020.igem.org/Team%3AOUC-China/Model#model_sd)
  + [Software](https://2020.igem.org/Team%3AOUC-China/Software)
* [PARTS](https://2020.igem.org/Team%3AOUC-China/Parts)
  + [Overview](https://2020.igem.org/Team%3AOUC-China/Parts)
  + [Improvement](https://2020.igem.org/Team%3AOUC-China/Parts#part_impro)
  + [Collection](https://2020.igem.org/Team%3AOUC-China/Part_Collection)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AOUC-China/Human_Practices)
  + [Human Practices](https://2020.igem.org/Team%3AOUC-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AOUC-China/Education)
  + [Comics](https://2020.igem.org/Team%3AOUC-China/Comics)
* [COLLABORATIONS](https://2020.igem.org/Team%3AOUC-China/Collaborations)
  + [Collaborations](https://2020.igem.org/Team%3AOUC-China/Collaborations)
  + [Meet Up](https://2020.igem.org/Team%3AOUC-China/Collaborations#col_mu)
  + [Team List](https://2020.igem.org/Team%3AOUC-China/Collaborations#col_tl)
  + [Partnership](https://2020.igem.org/Team%3AOUC-China/Partnership)
* [TEAM](https://2020.igem.org/Team%3AOUC-China/Team)
  + [Members](https://2020.igem.org/Team%3AOUC-China/Team)
  + [Attributions](https://2020.igem.org/Team%3AOUC-China/Attributions)

[![](https://static.igem.org/mediawiki/2020/0/0a/T--OUC-China--back_to_top.png)](#toptop)

![](https://static.igem.org/mediawiki/2020/7/75/T--OUC-China--protocol_banner.jpg)

This year, we use the following protocols to complete our experiments. And we have sorted out them in detail.

# 1. Elementary Operation

[(1) Conservation](http://dx.doi.org/10.17504/protocols.io.bnb7marn)

[(2) Semi-solid Agar Stab Strain Preservation Method](http://dx.doi.org/10.17504/protocols.io.bnhvmb66)

[(3) Inoculation](http://dx.doi.org/10.17504/protocols.io.bndkma4w)

[(4) Preparative Methods Of Competent Escherichia coli Cells](http://dx.doi.org/10.17504/protocols.io.bndmma46)

[(5) Plasmid Extraction](http://dx.doi.org/10.17504/protocols.io.bndrma56)

[(6) Transformation Of Escherichia coli](http://dx.doi.org/10.17504/protocols.io.bndpma5n)

[(7) DNA Purification](http://dx.doi.org/10.17504/protocols.io.bndtma6n)

[(8) Fluorescence Measurement](http://dx.doi.org/10.17504/protocols.io.bndqma5w)

# 2. Demonstration

[(1) Colony Polymerase Chain Reaction](http://dx.doi.org/10.17504/protocols.io.bndsma6e)

[(2) Gel Electrophoresis](http://dx.doi.org/10.17504/protocols.io.bndnma5e)

[(3) Gel Extraction](http://dx.doi.org/10.17504/protocols.io.bnduma6w)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:OUC-China/Results

[![](https://static.igem.org/mediawiki/2020/7/73/T--OUC-China--middle_button_on.png)](https://2020.igem.org/Team%3AOUC-China)

* PROJECT
  + [Description](https://2020.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2020.igem.org/Team%3AOUC-China/Design)
  + [Results](https://2020.igem.org/Team%3AOUC-China/Results)
  + [Contribution](https://2020.igem.org/Team%3AOUC-China/Contribution)
  + [Engineering](https://2020.igem.org/Team%3AOUC-China/Engineering)
  + [Implementation](https://2020.igem.org/Team%3AOUC-China/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3AOUC-China/Proof_Of_Concept)
* WET LAB
  + [Experiments](https://2020.igem.org/Team%3AOUC-China/Experiments)
  + [Safety](https://2020.igem.org/Team%3AOUC-China/Safety)
  + [Measurement](https://2020.igem.org/Team%3AOUC-China/Measurement)
* [MODEL](https://2020.igem.org/Team%3AOUC-China/Model)
  + [Overview](https://2020.igem.org/Team%3AOUC-China/Model#model_ov_po)
  + [3WJ](https://2020.igem.org/Team%3AOUC-China/Model#model_3wj)
  + [ODE](https://2020.igem.org/Team%3AOUC-China/Model#model_ode)
  + [Sequence Design](https://2020.igem.org/Team%3AOUC-China/Model#model_sd)
  + [Software](https://2020.igem.org/Team%3AOUC-China/Software)
* [PARTS](https://2020.igem.org/Team%3AOUC-China/Parts)
  + [Overview](https://2020.igem.org/Team%3AOUC-China/Parts)
  + [Improvement](https://2020.igem.org/Team%3AOUC-China/Parts#part_impro)
  + [Collection](https://2020.igem.org/Team%3AOUC-China/Part_Collection)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AOUC-China/Human_Practices)
  + [Human Practices](https://2020.igem.org/Team%3AOUC-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AOUC-China/Education)
  + [Comics](https://2020.igem.org/Team%3AOUC-China/Comics)
* [COLLABORATIONS](https://2020.igem.org/Team%3AOUC-China/Collaborations)
  + [Collaborations](https://2020.igem.org/Team%3AOUC-China/Collaborations)
  + [Meet Up](https://2020.igem.org/Team%3AOUC-China/Collaborations#col_mu)
  + [Team List](https://2020.igem.org/Team%3AOUC-China/Collaborations#col_tl)
  + [Partnership](https://2020.igem.org/Team%3AOUC-China/Partnership)
* [TEAM](https://2020.igem.org/Team%3AOUC-China/Team)
  + [Members](https://2020.igem.org/Team%3AOUC-China/Team)
  + [Attributions](https://2020.igem.org/Team%3AOUC-China/Attributions)

[![](https://static.igem.org/mediawiki/2020/0/0a/T--OUC-China--back_to_top.png)](#toptop)

![](https://static.igem.org/mediawiki/2020/f/f2/T--OUC-China--result_banner.jpg)

[Preparations](#result_prepa)

[Logic Gates](#result_lg)

[OR Gate](#result_lg_or)

[AND Gate](#result_lg_and)

[NOT Gate](#result_lg_not)

[NIMPLY Gate](#result_lg_nimply)

[IMPLY Gate](#result_lg_imply)

[XOR Gate](#result_lg_xor)

[Improvements](#result_improve)

[Stability](#result_im_sta)

[Versatility](#result_im_ver)

[Composability](#result_im_com)

[Multiple-input](#result_im_mul)

[Swapping Gates](#result_im_swa)

[Application](#result_appli)

[Arithmetic Units](#result_appli_au)

[Virus Detection](#result_appli_vd)

[Future](#result_fut)

How can we build a biological computer? Since the basic functional unit of an electronic computer is implemented through logic gates, we think that we can start by optimizing and perfecting the logic gate. We incorporated a series of logic gates which were built by toehold and 3WJ repressor, and designed the IMPLY gate and XOR gate that are still devoid. We made some improvements to these logic gates to improve their stability, versatility, composability, and designed multiple-input logic gates and swapping gates. In order to show the wide application prospect of these logic gates, we used them in the construction of adder and subtractor, as well as virus detection. We will introduce our results in the following aspects: preparations, logic gates, improvements and applications.

# 1. Preparations

In order to build logic gates better, we need to do some preparations. Therefore, we have conducted a series of tests on promoters, the ON RNA switch and the OFF RNA switch. As we introduced in [DESIGN](https://2020.igem.org/Team%3AOUC-China/Design), we chose toehold as the ON RNA switch and three-way Junction (3WJ) repressor as the OFF RNA switch. The results of these basic tests provided references for the construction of logic gates and the design of some more complex circuits.

## 1.1 Promoters

It is very important for us to select the inducible promoter with low transcriptional leakage and high dynamic range. These promoters are essential parts of building the logic gates. These promoters also played a crucial role in the subsequent construction of multiple-input logic gates and swapping gates. After a series of experimental tests, we selected Tet promoter and Lux promoter with good performance to build the double-input logic gate. Affected by COVID-19, we don’t have enough time to test all promoters, so we obtained the experimental data of the pBAD from the NEFU\_China.

|  |  |
| --- | --- |
| ![](https://static.igem.org/mediawiki/2020/8/86/T--OUC-China--result_fig1A_pTet.jpg) | ![](https://static.igem.org/mediawiki/2020/d/d5/T--OUC-China--result_fig1B_pLux.jpg) |

Figure 1. The fluorescence data of promoters.

(A) The Tet promoter fluorescence data in E. coli BL21 (DE3) at different aTc concentrations (0mg/mL, 0.01mg/mL, 0.1mg/mL, 0.25mg/mL, 0.5mg/mL and 1mg/mL). The Tet promoter showed the highest fluorescence intensity when the aTc concentration was 0.25mg/mL. (B) The Lux promoter fluorescence data in E. coli BL21 (DE3) at different HSL (N-(Ketocaproyl)-L-homoserine Lactone) concentrations (0mg/mL, 0.001mg/mL, 0.002mg/mL, 0.01mg/mL, 0.1mg/mL and 1mg/mL). The Lux promoter showed the highest fluorescence intensity when the HSL concentration was 0.01mg/mL. Error bar: SD (n=9).

We confirmed the optimal concentration of inducers for Tet promoter and Lux promoter. The Tet promoter showed the highest fluorescence intensity when the aTc concentration was 0.25mg/mL, and the Lux promoter showed the highest fluorescence intensity when the HSL (N-(Ketocaproyl)-L-homoserine Lactone) concentration was 0.01mg/mL. In subsequent experiments, the optimal concentration of inducers was used to being inputs to logic gates, in addition to verify the performance of toehold and 3WJ repressor.

## 1.2 Toehold

Toehold switch is composed of cis-acting element RNA hairpins and trans-acting factor trigger RNA. The binding of a trigger RNA to the toehold sequence allows for a branch migration process, exposing AUG and RBS for translation initiation.

![](https://static.igem.org/mediawiki/2020/7/7e/T--OUC-China--result_fig2_th.jpg)

Figure 2. Validation of toehold

Compared with the blank control (IPTG=0 M, aTc=0 mg/mL), the fluorescence of GFP was low when only the promoter before toehold sequence was turned on (IPTG=0.1 M, aTc=0 mg/mL). This indicates that toehold has the advantage of low leakage. When the trigger was expressed (IPTG=0.1 M, aTc=0.25 mg/mL), it showed a high GFP fluorescence of up to 32-fold due to the destruction of the toehold hairpin structure. Error bar: SD (n=9).

Because toehold has the characteristics of low leakage and high ON/OFF ratio, we choose it as an ON switch. And we used it in the construction of logic gates and the design of some more complex circuits.

## 1.3 Three-way Junction (3WJ) Repressor

3WJ repressor switch RNA employs an unstable hairpin secondary structure. This unstable hairpin was previously demonstrated to be translationally active. When a complementary trigger RNA is expressed, the trigger will bind to the switch RNA, making the originally unstable 3WJ structure stable, and represses translation.

![](https://static.igem.org/mediawiki/2020/c/c6/T--OUC-China--result_fig3.jpg)

Figure 3. The inhibitory effect of 3WJ repressors

In the validation of 3WJ, as with toehold, we also set blank control (IPTG=0 M, aTc=0 mg/mL). Compared with the group without trigger expression (IPTG=0.1 M, aTc=0 mg/mL), the group with trigger expression (IPTG=0.1 M, aTc=0.25 mg/mL) showed inhibitory effect. Error bar: SD (n=9).

![](https://static.igem.org/mediawiki/2020/3/31/T--OUC-China--result_fig4.jpg)

Figure 4. Orthogonality of 3WJ repressors

Crosstalk was determined by dividing the arithmetic mean of the GFP fluorescence from a given trigger switch pair by the arithmetic mean of the GFP fluorescence for the cognate trigger switch interaction. GFP fluorescence was measured from n=9 biologically independent samples.

3WJ repressor showed remarkable ON/OFF ratio and had good orthogonality, we choose it as OFF switch. And we used it in the construction of logic gates and the design of some more complex circuits.

# 2. Logic Gates

In the verification of the logic circuit, the inductors corresponding to the promoter before the switch structure were taken as the inputs (after adding aTc, trigger A will be expressed and after adding HSL, trigger B will be expressed), and the expression quantity of fluorescent protein was taken as the output, so as to test the existing logic operations and the logic operations designed by us (IMPLY gate and XOR gate).

## 2.1 OR Gate

OR gate composed of two switch RNA hairpins. Each switch module has an input RNA recognition site and its own RBS and start codon. Input RNA binding unwinds the corresponding switch stem to activate translation.

![](https://static.igem.org/mediawiki/2020/1/19/T--OUC-China--result_fig5.jpg)

Figure 5. Two-input toehold OR gate

The left figure shows that when INPUT A=0, INPUT B=0, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).

## 2.2 AND Gate

AND gate constructed from two input RNAs that bind to yield a complete trigger RNA. When either input RNA is expressed, it is incapable of activating the switch because neither trigger sub-sequence alone can unwind the repressing hairpin. The toehold switch can only be turned on when the two input RNA species hybridize and form a complete trigger sequence.

![](https://static.igem.org/mediawiki/2020/4/4e/T--OUC-China--result_fig6.jpg)

Figure 6. Two-input toehold AND gate

The left figure shows that when INPUT A=1, INPUT B=1, the fluorescence of GFP is high. And the fluorescence intensity of GFP was low in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).

## 2.3 NOT Gate

NOT gate is made up of 3WJ repressor. Its unstable hairpin was previously demonstrated to be translationally active. When a complementary trigger RNA is expressed, the trigger will bind to the switch RNA, making the originally unstable 3WJ structure stable, and represses translation.

![](https://static.igem.org/mediawiki/2020/4/4b/T--OUC-China--result_fig7.jpg)

Figure 7. Two-input 3WJ repressor NOT gate

The left figure shows that when INPUT=0, the fluorescence of GFP is high. And the fluorescence intensity of GFP was low when INPUT=1. This corresponds to the situation described in the truth table on the right. INPUT=1 means that aTc (0.25 mg/mL) is added. Error bar: SD (n=9).

## 2.4 NIMPLY Gate

In the NIMPLY gate, a deactivating RNA (INPUT A) uses direct hybridization or strand displacement to abolish trigger RNA (input B) activity. So when only the correct trigger RNA is expressed, the switch can be turned on.

![](https://static.igem.org/mediawiki/2020/7/7e/T--OUC-China--result_fig8.jpg)

Figure 8. Two-input 3WJ repressor NIMPLY gate

The left figure shows that when INPUT A=0, INPUT B=1, the fluorescence of GFP is high. And the fluorescence intensity of GFP was low in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).

## 2.5 IMPLY Gate

We combined the 3WJ switch and toehold switch to realize the IMPLY Boolean calculation. When no trigger is expressed, this logic gate just likes a 3WJ switch. When trigger A expressed, the trigger will bind to the switch RNA. The binding allows for a branch migration process, exposing AUG and RBS for translation initiation. When trigger B expressed, the trigger will bind to 3WJ switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and the start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation. When both trigger A and B are expressed, the binding of trigger RNA to the toehold sequence allows the ribosome to bind to the former RBS and break open 3WJ stable hairpin.

![](https://static.igem.org/mediawiki/2020/8/85/T--OUC-China--result_fig9.jpg)

Figure 9. Two-input toehold and 3WJ repressor IMPLY gate

The left figure shows that when INPUT A=0, INPUT B=1, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).

## 2.6 XOR Gate

The XOR is inspired by the NIMPLY gate, consisting of a toehold switch and two triggers. The trigger’s core sequence is the same and at the triggers’ both ends there are the nucleotide-binding domains. When input one of these triggers, the switch can turn on. Moreover, when input these two triggers simultaneously, they can pair together and form a ring in the middle. As a result, the switch will still be in the OFF state.

![](https://static.igem.org/mediawiki/2020/f/f3/T--OUC-China--result_fig10.jpg)

Figure 10. Two-input toehold XOR gate

The left figure shows that when INPUT A and INPUT B are both 0 or 1, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other two groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).

# 3. Improvements

Stability, versatility and compatibility are important for biocomputers. We add the hairpin structure at the 5' end of triggers to improve the trigger stability, design the software for calculating the threshold to improve the universality, and use trans-action factors such as σ factor to improve the compatibility. We also designed more complex multiple-input logic gates and swapping gates to show more possibilities of logic gates.

## 3.1 Stability

The stability of RNA is a key factor affecting the mechanism of its interaction. Therefore, we hope to optimize the ON/OFF ratio of the toehold switch by improving the stability of trigger RNA. After consulting many pieces of literature, we learned that adding hairpin structure at the 5 'end of RNA could prevent the degradation mediated by RNase, thus improving the stability of RNA. Therefore, we tried to add the hairpin structure at the 5' end of triggers. We used NUPACK and RNAfold to analyze the structure of the trigger with the 5’ end hairpin, and then used NUPACK to analyze the combination of triggers with the 5’ end hairpin and switches.

![](https://static.igem.org/mediawiki/2020/b/b2/T--OUC-China--result_fig11.jpg)

Figure 11. The secondary structure

(A) The secondary structure of 5’ hairpins analyzed using NUPACK. (B) The secondary structure of 5’ hairpins combining with trigger analyzed using NUPACK.

## 3.2 Versatility

We find a certain difference in the threshold value of different logic gates, which is not conducive to combining different types of logic gates. To improve the generality of these logic gates, it is necessary to find a universal threshold. Therefore, we have developed a [SOFTWARE](https://2020.igem.org/Team%3AOUC-China/Software) that can calculate the common threshold. Threshold calculation can give the normalized parameters of fluorescence intensity and the calculated threshold, so that the designers can get the common threshold of different logic gates easily and combine different types of logic gates better.

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| ![](https://static.igem.org/mediawiki/2020/f/fa/T--OUC-China--result_fig12_1.jpg) | ![](https://static.igem.org/mediawiki/2020/5/55/T--OUC-China--result_fig12_2.jpg) |

Figure 12. Demonstration of software

![](https://static.igem.org/mediawiki/2020/7/7d/T--OUC-China--result_fig13.png)

Figure 13. The normalization of date

## 3.3 Composability

Incorporation of ribo-computing devices into sophisticated layered circuits will require systems that can provide RNAs as output species. Thus, we use σ factors to achieve this function. The σ factors have several highly orthogonal pairs, so that this functionality can be implemented.

## 3.4 Multiple-input

On the basis of single-input logic gates and two-input logic gates, we also designed the three-input logic gates and four-input logic gates. These enrich the types of our logic gates. See the [MODEL (Multiple-input)](https://2020.igem.org/Team%3AOUC-China/Model#model_sd_multi) page for details.

![](https://static.igem.org/mediawiki/2020/b/bf/T--OUC-China--result_fig14.jpg)

Figure 14. The secondary structure of the three-inputs logic gates

![](https://static.igem.org/mediawiki/2020/1/14/T--OUC-China--result_fig15.jpg)

Figure 15. The secondary structure of the four-inputs logic gates

## 3.5 Swapping Gates

In the IMPLY and NOT swapping gate, we use a trigger control the transformation between two logic gates. We combined the inhibitory hairpin constructed switch with the 3WJ repressor, which achieved the transformation between IMPLY and NOT. If input the trigger A, it is IMPLY gate. If not, it is NOT gate.

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| ![](https://static.igem.org/mediawiki/2020/4/4d/T--OUC-China--result_fig16A.png) | ![](https://static.igem.org/mediawiki/2020/5/56/T--OUC-China--result_fig16B.png) |

Figure 16. The secondary structure of IMPLY and NOT swapping gate

In the AND and OR swapping gate, we use different triggers to control the transformation. We combined the single hairpin constructed switch and the inhibitory hairpin constructed switch to achieve the transformation between AND gate and OR gate. When input the trigger B, it is OR gate. When trigger A is absent, it behaves like an AND gate.

![](https://static.igem.org/mediawiki/2020/7/78/T--OUC-China--result_fig17.png)

Figure 17. The secondary structure of AND and OR swapping gate

# 4. Application

As a basic logic gate element, our project can be applied to many aspects. In order to show the wide application prospect of these logic gates, we used them in the construction of half-adder and subtractor, as well as virus detection.

## 4.1 Arithmetic Units

We construct the half-adder and the subtractor with the AND, XOR and NIMPLY gates. The half-adder is capable of carrying out simple binary mathematical operations necessary for digital computing. The subtractor can be done by adjusting the concentration of trigger RNAs. In the future, they can be used in the more complicated calculations.

### 4.1.1 Half-adder

We used the AND gate and XOR gate to make up the half-adder. A half adder is a type of adder, an electronic circuit that performs the addition of numbers. The half adder is able to add two single binary digits and provide the output plus a carrying value. It has two inputs, called A and B, and two outputs S (sum) and C (carry).

The XOR is inspired by the NIMPLY gate, consisting of a toehold switch and two triggers. The trigger’s core sequence is the same and at the triggers’ both ends there are the nucleotide-binding domains. When input one of these triggers, the switch can turn on. Moreover, when input these two triggers simultaneously, they can pair together and form a ring in the middle. As a result, the switch will still be in the OFF state.

AND gate constructed from two input RNAs that bind to yield a complete trigger RNA. When either input RNA is expressed, it is incapable of activating the switch because neither trigger sub-sequence alone can unwind the repressing hairpin. The toehold switch can only be turned on when the two input RNA species hybridize and form a complete trigger sequence.

When there is only one input, the reporter of AND gate is not expressed, and the reporter of XOR gate is expressed. When there are two inputs, the reporter of AND gate is expressed, and the reporter of XOR gate is not expressed.

![](https://static.igem.org/mediawiki/2020/9/92/T--OUC-China--result_fig18.jpg)

Figure 18. Half-adder circuit diagram

### 4.1.2 Subtractor

We used the NIMPLY gate to make up the subtractor. We use inducers to turn on trigger expression, use inducer concentration to represent the input trigger concentration, and use fluorescent protein expression levels to react and output results. When two triggers are expressed at the same time, trigger S1 and trigger S2 will combine to form an RNA double strand that cannot be turned on by RNA Switch. Only when trigger S2 is expressed, the downstream gene expression of RNA Switch will be turned on. This subtracter can simulate S2-S1=Ps, and Ps is the fluorescence intensity of the reporter gene.

![](https://static.igem.org/mediawiki/2020/1/18/T--OUC-China--result_subtracter.jpg)

Figure 19. Abstractions of subtractor

However, we only find one NIMPLY gate sequences in the literature. In order to achieve the subtractor that can calculate different concentration range, we designed NIMPLY upon that sequence.

The NIMPLY gate consists of a toehold switch and two input triggers that can pair together. The toehold and the triggers’ core sequences we used originated from the previous literature. Then we added the nucleotide-binding domains at both ends of the trigger’s core sequence. This sequence is named “trigger1”. The other one was designed for completely complementary pairing with trigger1 and not pairing with switch sequence. These sequences were designed using NUPACK and were further screened using RNAfold and RNAstructure.

![](https://static.igem.org/mediawiki/2020/6/65/T--OUC-China--result_fig19.png)

Figure 20. The secondary structure of the pairing two triggers analyzed using NUPACK.

## 4.2 Virus Detection

Under the influence of COVID-19, our experimental time was more limited and the control of virus usage became stricter. Therefore, we didn’t conduct experimental verification on the application of virus detection, but only demonstrated the application of virus detection with a logic gate in a biological computer through design and modeling simulation.

# 5. Future

The ultimate goal of combining molecular biology and engineering is to realize the biocomputer. Since an electronic computer is so well-developed according to our research, it seemed unlikely that the biocomputer element we research into will easily surpass the calculation speed of electronic computers. Therefore, biocomputer should demonstrate excellence in another area, such as, high humidity tolerance, high biocompatibility and mass storage capacity. Biocomputer should have special functions which emphasis on biological characteristic and would be complementary with electronic computers eventually. The expertise we obtained has inspired us to consider realizing biological functions with our biocomputer designs.

Our project is not trying to solve a real world problem with practical applications right away, we build elements of a biological computer. Logic gates are the basis of computing. To build a biocomputer, we need to start from the basic parts built by biological elements. We believe that these logic gates have great potential in resistance gene detection, virus detection, and disease detection.

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