iGEM 2020 — Team SUSTech_Shenzhen

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# Team:SUSTech Shenzhen/Notebook

Notebook – Team:SUSTech Shenzhen

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## Notebook

details of our experiments

## Stage 1. Virtual Screening

![](https://static.igem.org/mediawiki/2020/7/72/T--SUSTech_Shenzhen--1-11.png)

![](https://static.igem.org/mediawiki/2020/c/cb/T--SUSTech_Shenzhen--3-7.png)

![](https://static.igem.org/mediawiki/2020/c/c1/T--SUSTech_Shenzhen--2-10.png)

![](https://static.igem.org/mediawiki/2020/5/5c/T--SUSTech_Shenzhen--4-3.png)

![](https://static.igem.org/mediawiki/2020/3/32/T--SUSTech_Shenzhen--Q839.png)

![](https://static.igem.org/mediawiki/2020/0/0f/T--SUSTech_Shenzhen--5-4.png)

![](https://static.igem.org/mediawiki/2020/0/0f/T--SUSTech_Shenzhen--6-3.png)

![](https://static.igem.org/mediawiki/2020/e/ee/T--SUSTech_Shenzhen--7-3.png)

![](https://static.igem.org/mediawiki/2020/c/cb/T--SUSTech_Shenzhen--8-1.png)

**6.20-6.23**:
Screening over the first chemical library (800
candidates)

**6.24-5.30**:
Screening over the Second chemical library (2,000
candidates)

**7.10**:
Optimize the Condition for DSF assay (three factors
including protein concentration, dye concentration, and
buffer type)

**7.14-7.20**:
Use different Concentrations of protein (5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60 uM) with 2.5x SYPRO red
dye (final concentration) and found out that 50 uM
concentration was suitable for detection.

**8.1-8.10**:
Use different concentrations of fluorescence dye (SYPRO
red) to optimize the detection of protein stability
shift using 100 uM DNA as a positive control, and found
out that 12.5x final concentration is the best.

**8.16-8.30**:
Purchase the top 30 chemical candidates and test them
with the system described below and search for possible
candidates.

## Stage 2. Report System Constructing

![](https://static.igem.org/mediawiki/2020/8/8d/T--SUSTech_Shenzhen--160.jpg)

![](https://static.igem.org/mediawiki/2020/f/fc/T--SUSTech_Shenzhen--IMG_3430.jpg)

![](https://static.igem.org/mediawiki/2020/d/d9/T--SUSTech_Shenzhen--IMG_1230.jpg)

![](https://static.igem.org/mediawiki/2020/f/f5/T--SUSTech_Shenzhen--IMG_1231.jpg)

![](https://static.igem.org/mediawiki/2020/1/14/T--SUSTech_Shenzhen--IMG_1234.jpg)

![](https://static.igem.org/mediawiki/2020/c/ca/T--SUSTech_Shenzhen--IMG_1239.jpg)

![](https://static.igem.org/mediawiki/2020/4/48/T--SUSTech_Shenzhen--IMG_1240.jpg)

![](https://static.igem.org/mediawiki/2020/c/c3/T--SUSTech_Shenzhen--IMG_1244.jpg)

![](https://static.igem.org/mediawiki/2020/1/14/T--SUSTech_Shenzhen--IMG_1308.jpg)

**8.13**:
Amplify the GFP fragment with PCR (EcoRI and HindIII
sites were added by the primers).

**8.16**:
Enzyme EcoRI and HindIII were used to cut the amplified
fragment and plasmid pUCP20 from Yang Lab, the processed
two parts were then ligated by ligase.

**8.17**:
Plasmid pUCP20-GFP was amplified by DH5α, and primer M13
fwd was used for the sequencing of the GFP region.

**8.18**: The
promoter region of *acr was* synthesized by IDT,
the origin lac promoter of pUCP20-GFP was replaced by
the acr promoter using homologous recombination.

**8.19**:
Plasmid pUCP20-GFP-acr promoter was transformed into
BL21-CodonPlus (DE3)-RIL to test the efficiency of the
acr promoter. BL21-CodonPlus (DE3)-RIL turned green.

**8.20-8.25**:
Gene *aca1* was added to pRSFDuet-1 using
homologous recombination. (*aca1* was synthesized
by IDT).

**8.26-8.30**:
Plasmids pRSFDuet-1-aca1 and pUCP20-GFP-acr promoter
were co-transformed into BL21-CodonPlus (DE3)-RIL. With
the presence of IPTG, which can induce the expression of
Aca1, the E.Coli was not as green as that without IPTG.

## Stage 3. Test the Candidate Compounds by the Report System

![](https://static.igem.org/mediawiki/2020/2/2f/T--SUSTech_Shenzhen--IMG_3442.jpg)

![](https://static.igem.org/mediawiki/2020/a/ac/T--SUSTech_Shenzhen--IMG_1250.jpg)

![](https://static.igem.org/mediawiki/2020/8/8c/T--SUSTech_Shenzhen--IMG_1251.jpg)

![](https://static.igem.org/mediawiki/2020/f/f3/T--SUSTech_Shenzhen--IMG_1252.jpg)

![](https://static.igem.org/mediawiki/2020/f/f7/T--SUSTech_Shenzhen--IMG_12525.jpg)

![](https://static.igem.org/mediawiki/2020/4/4c/T--SUSTech_Shenzhen--IMG_1255.jpg)

![](https://static.igem.org/mediawiki/2020/b/be/T--SUSTech_Shenzhen--IMG_1256.jpg)

![](https://static.igem.org/mediawiki/2020/2/25/T--SUSTech_Shenzhen--IMG_1258.jpg)

![](https://static.igem.org/mediawiki/2020/4/4d/T--SUSTech_Shenzhen--IMG_1302.jpg)

**9.1-9.5**:
BL21-CodonPlus (DE3)-RIL containing plasmids
pRSFDuet-1-aca1(pRSFDuet-1-aca1R44A for positive
control) and the pUCP20-GFP-acr promoter was cultured in
LB to OD600 =0.5 with 500uM IPTG, then the
candidate compounds were added separately to 105ul
bacteria liquid (concentration of compounds are 39uM).
After overnight growth at 37oC 220 rpm, green
fluorescence was observed with a microplate reader.
Select those who have the closest green fluorescence to
the aca1R44A one.

**9.6-9.10**:
Increase the concentration of the selected compounds and
repeat the experiments.

## Stage 4. Confirm the Selected compound with DSF

![](https://static.igem.org/mediawiki/2020/5/53/T--SUSTech_Shenzhen--IMG_3425.jpg)

![](https://static.igem.org/mediawiki/2020/7/7f/T--SUSTech_Shenzhen--IMG_3426.jpg)

![](https://static.igem.org/mediawiki/2020/5/57/T--SUSTech_Shenzhen--IMG_3427.jpg)

![](https://static.igem.org/mediawiki/2020/7/70/T--SUSTech_Shenzhen--IMG_3432.jpg)

![](https://static.igem.org/mediawiki/2020/7/73/T--SUSTech_Shenzhen--IMG_3434.jpg)

![](https://static.igem.org/mediawiki/2020/f/fb/T--SUSTech_Shenzhen--IMG_3435.jpg)

![](https://static.igem.org/mediawiki/2020/7/7b/T--SUSTech_Shenzhen--IMG_3436.jpg)

![](https://static.igem.org/mediawiki/2020/a/ae/T--SUSTech_Shenzhen--IMG_3441.jpg)

![](https://static.igem.org/mediawiki/2020/2/2b/T--SUSTech_Shenzhen--IMG_1237.jpg)

**10.10-10.15**:
DSF was used to detect the binding of the compound and
Aca1.

**10.16-10.22**:
Designed 60 chemically modified versions of our chemical
candidate, and used *Molecular Operating
Environment* to estimated the strength of
protein-ligand interaction.

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@iGEM\_SUSTech

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# Team:SUSTech Shenzhen/Engineering

Engineering – Team:SUSTech Shenzhen

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Team:SUSTech Shenzhen

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## Engineering

how we succeed in the engineering

## Overall Engineering Success

Through the literature review, we found that
Aca1 protein was important in the survival of *Pseudomonas*
phages1. Such protection depends on the interaction between
Aca1 protein and a promoter (*acr*-promoter) that regulates the
expression of *acr* gene1. Therefore, we developed an
idea that we could disrupt the binding between Aca1 protein and
*acr*-promoter to induce the death of *Pseudomonas*
phages. We expected such disruption was carried out by a drug, a small
molecule. To develop such a drug, we designed a screening system to
firstly find a potential inhibitor and then optimize it. Such a system
was built in our experiment.

![](https://static.igem.org/mediawiki/2020/4/4c/T--SUSTech_Shenzhen--1-3-1024.png)

Figure 1. The workflow of our screening
system

In this screening system, we firstly
screened inhibitor candidates in our virtual library. And then, for
those inhibitor candidates, we tested if they were functional as our
expectation in our cellular screening system built-in *E. coli*.
For the ones that resulted in we expect results, we were going to
further demonstrate their binding with Aca1 via biochemical approaches
in order to avoid false positives. As such potential inhibitors may not
reach the highest efficiency, after biochemical validation we planned to
optimize them via chemical access. For those optimized inhibitors, they
would be tested in *Pseudomonas* to find out if it could really
defend against the infection of phages. Finally, the ones that succeeded
in defending phage infection of *Pseudomonas* were the drugs we
wanted.

## Engineering Success in E. coli validation system

In our *E. coli* validation system,
we expected to successfully screen our inhibitor candidates with a
system simulating phage infected *Pseudomonas*. In such a system,
we could screen for effective inhibitors for the binding between Aca1
and *acr*-promoter. We got an idea to use the expression level of
eGFP2, a fluorescence protein, to reflect the activity of
*acr*-promoter. That is to say, in our system, a stronger
fluorescence illuminance could reflect a higher activity of *acr*-promoter.
As Aca1 protein could bind to *acr*-promoter and inactivate
it1, we expected a decrease in fluorescence illuminance with
the presence of Aca1 protein and recovery of fluorescence illuminance
after an effective inhibitor was added.

Therefore, the challenge we faced in
building such a validation system was how to design functional plasmid
to realize a simulation system in *E. coli*. We wanted to achieve
a successful system by designing plasmids. Then, we were going to test
the feasibility of such a plasmid system for the expected lowering of
fluorescence illuminance in the presence of Aca1. If it turned out that
such a plasmid system worked, we would then use such a system to screen
our inhibitor candidates with the [**model**](https://2020.igem.org/Team%3ASUSTech_Shenzhen/Model) we built. Finally,
the one that turned out to have the highest efficiency was what we
wanted.

![](https://static.igem.org/mediawiki/2020/7/70/T--SUSTech_Shenzhen--2-1-1024.png)

Figure 2. The workflow of building *E.
coli* validation system

In the first attempt, we build a plasmid
that was expected to carry all the important information in our expected
system. On this plasmid, *acr*-promoter regulated the
transcription of *egfp* gene. And *aca1* gene was also
located on this plasmid under the regulation of *lacI* promoter
to ensure a steady high transcription level. However, after transforming
such plasmid into *E. coli*, we found that the fluorescence
illuminance in such *E. coli* is the same as the *E. coli*
transformed with a plasmid without any *aca1* gene. Such results
indicated that the *aca1* gene in such a system failed to be
expressed. Therefore, this system could not work as our expectation and
we tried another way to design plasmids.

![](https://static.igem.org/mediawiki/2020/7/7b/T--SUSTech_Shenzhen--3-1-1024.png)

Figure 3. Illustration of plasmid design at
the first attempt

In the second attempt, we turned to create a
two-plasmid system, one plasmid for Aca1 expression and the other one
for reporting. In this way, we engineered the plasmid pUCP20 (*Pseudomonas* plasmid)
by inserting the report gene *egfp* after the *acr*-promoter.
And *aca1* gene was inserted after *lac* operon in the
plasmid pRSFduet. Then, the engineering pUPC20 and pRSF1b plasmids were
co-transformed into*E. coli* (BL21DE3). Due to the
different antibiotic-resistant genes the two plasmids had, we readily
selected the *E. coli* which successfully
co-transformed by using ampicillin and kanamycin. And we found that
such a system just worked as our expectation.

![](https://static.igem.org/mediawiki/2020/b/bb/T--SUSTech_Shenzhen--4.jpg)

Figure 4. Engineering pUCP20 plasmid

![](https://static.igem.org/mediawiki/2020/f/f5/T--SUSTech_Shenzhen--5-1.jpg)

Figure 5. Engineering pRSFduet plasmid

Finally, using the two-plasmid system (a
composite Part: [BBa\_K3423005](http://parts.igem.org/Part%3ABBa_K3423005)
made up of [BBa\_E0040](http://parts.igem.org/Part%3ABBa_BBa_E0040),
[BBa\_J61104](http://parts.igem.org/Part%3ABBa_BBa_J61104), [BBa\_I719005](http://parts.igem.org/Part%3ABBa_BBa_I719005),
[BBa\_B0012](http://parts.igem.org/Part%3ABBa_BBa_B0012), [BBa\_K3423000](http://parts.igem.org/Part%3ABBa_BBa_K3423000),
and
[BBa\_K3423001](http://parts.igem.org/Part%3ABBa_BBa_K3423001))we
developed at our second attempt, we succeeded in screening potential
inhibitors among our inhibitor candidates with our [model](https://2020.igem.org/Team%3ASUSTech_Shenzhen/Model).

Table 1. *in vivo*
*KI* of different PDI candidates

| **compounds** | ***c(PDI-inhibitor)* (μM)** | **I0** | **I** | **I1** | **KI (μM)** |
| --- | --- | --- | --- | --- | --- |
| 1388-F10 | 19 | 167401 | 191357 | 1446723 | 115.2079 |
| 1388-B9 | 19 | 167401 | 192753 | 1446723 | 108.743 |
| 1401-H11 | 19 | 167401 | 220510.3 | 1446723 | 50.75995 |
| 1401-G2 | 19 | 167401 | 222787 | 1446723 | 48.58307 |
| 1402-C10 | 19 | 167401 | 227404.7 | 1446723 | 44.6751 |
| 1388-D4 | 19 | 167401 | 228061.7 | 1446723 | 44.16743 |
| 1388-H6 | 19 | 167401 | 237461.3 | 1446723 | 37.94673 |
| 1405-H10 | 19 | 167401 | 271971.7 | 1446723 | 24.69803 |
| 1389-B10 | 19 | 167401 | 87026.33 | 1446723 | -37.192 |
| 1400-C4 | 19 | 167401 | 146922 | 1446723 | -139.539 |

![](https://static.igem.org/mediawiki/2020/d/dc/T--SUSTech_Shenzhen--6.png)

Figure 6. *in vivo* KI of
different PDI candidates

## References

1. Stanley, S. Y., *et al.* Anti-CRISPR-Associated Proteins Are
   Crucial Repressors of Anti-CRISPR Transcription. *Cell*
   **178**, 1452-1464.e1413,
   doi:https://doi.org/10.1016/j.cell.2019.07.046 (2019).
2. Zhang, G., Gurtu, V. & Kain, S. R. An Enhanced Green Fluorescent
   Protein Allows Sensitive Detection of Gene Transfer in Mammalian
   Cells. *Biochemical and Biophysical Research Communications*
   **227**, 707-711, doi:10.1006/bbrc.1996.1573 (1996).

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# Team:SUSTech Shenzhen/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
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### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:SUSTech Shenzhen/Results

Results – Team:SUSTech Shenzhen

[跳转至内容](#content)

Team:SUSTech Shenzhen

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## Results

Wow~That’s amazing~

## Overview

Large-Scale fermentation using a densely-cultured bacterium that is utilized in industrial production often suffer severely from bacteriophage infection due to the lack of genetic diversity. Phage contamination is a worldwide problem in the fermentation industry. Here we present a chemical biology approach to developing a compound that can protect *Pseudomonas* from phages infection.

## Protein-ligand Docking

In silico screening was performed based on the Aca1 structure solved by our primary PI using the software Molecular Operating Environment (MOE) and several promising candidates were identified.

Table 1. In silico screening results

[![](https://static.igem.org/mediawiki/2020/9/9e/T--SUSTech_Shenzhen--1-6.png)](https://static.igem.org/mediawiki/2020/9/9e/T--SUSTech_Shenzhen--1-6.png)

## Report system constructing and in vivo screening

In the first plasmid, Aca1 is expressed by a promoter regulated by LacI. In the second, green fluorescent protein (GFP) is expressed by the Acr promoter, which is inhibited by Aca1 expressed by the first plasmid.

As when the critical binding site of protein Aca1 (the 44th amino acid) is mutated, Aca1 can no longer bind to the target DNA. We mutated the *aca1* gene in the first plasmid (the 44th­­­­ amino acid arginine is mutated to alanine) as a positive control.

With the presence of 500uM IPTG, Aca1 is overexpressed by the first plasmid to inhibit the*acr* promoter in the second plasmid. Thus, the expression of GFP inhibited. While for the positive control, as Aca1 is mutated to Aca1R44A, the *acr* promoter is not inhibited, and thus the expression level of GFP is higher than that of the wild type (WT) control. Though detecting the green fluorescence intensity, we can easily measure the expression level of the GFP and thus determine at what level *acr* promoter is inhibited. Due to the inhibition from Aca1, the green fluorescent intensity difference can be over 10 times, which proved the efficiency of this reporting system.

[![](https://static.igem.org/mediawiki/2020/4/42/T--SUSTech_Shenzhen--2-4-1024x411.png)](https://static.igem.org/mediawiki/2020/4/42/T--SUSTech_Shenzhen--2-4-1024x411.png)

Figure 1. The green fluorescent protein expressed by *acr* promoter in *E. coli*

We can detect green fluorescence from almost every *E. coli* cell, which means the transformation is successful and the report system is effective.

Then, 30 candidates we got form the virtual screening is tested, we applied our model to evaluate the efficiency of 10 different PDI inhibitor candidates and found that 1400-C4 has the smallest *in vivo KI* among those candidates. Therefore, we concluded that 1400-C4 could have the best inhibition efficiency and focused on 1400-C4 in the further experiment process.

Table 2. in vivo KI of different PDI candidates

| **compounds** | ***c(PDI-inhibitor)* (μM)** | **I0** | **I** | **I1** | **KI (μM)** |
| --- | --- | --- | --- | --- | --- |
| 1388-F10 | 19 | 167401 | 191357 | 1446723 | 115.2079 |
| 1388-B9 | 19 | 167401 | 192753 | 1446723 | 108.743 |
| 1401-H11 | 19 | 167401 | 220510.3 | 1446723 | 50.75995 |
| 1401-G2 | 19 | 167401 | 222787 | 1446723 | 48.58307 |
| 1402-C10 | 19 | 167401 | 227404.7 | 1446723 | 44.6751 |
| 1388-D4 | 19 | 167401 | 228061.7 | 1446723 | 44.16743 |
| 1388-H6 | 19 | 167401 | 237461.3 | 1446723 | 37.94673 |
| 1405-H10 | 19 | 167401 | 271971.7 | 1446723 | 24.69803 |
| 1389-B10 | 19 | 167401 | 87026.33 | 1446723 | -37.192 |
| 1400-C4 | 19 | 167401 | 146922 | 1446723 | -139.539 |

[![](https://static.igem.org/mediawiki/2020/d/dc/T--SUSTech_Shenzhen--6.png)](https://static.igem.org/mediawiki/2020/d/dc/T--SUSTech_Shenzhen--6.png)

Figure 2. *in vivo* KI of different PDI candidates

Then, we repeated several experiments using 1400-C4:

[![](https://static.igem.org/mediawiki/2020/6/66/T--SUSTech_Shenzhen--4-1-147.png)](https://static.igem.org/mediawiki/2020/2/2a/T--SUSTech_Shenzhen--4-1.png)

Figure 3. Efficiency of Navitoclax measured with green fluorescence intensity using part BBa\_K3423005

## Confirm the binding with differential scanning fluorimetry (DSF)

We confirmed the binding of the compound and Aca1 in vitro through DSF:

[![](https://static.igem.org/mediawiki/2020/9/9e/T--SUSTech_Shenzhen--5-1-1024.png)](https://static.igem.org/mediawiki/2020/9/9e/T--SUSTech_Shenzhen--5-1-1024.png)

[![](https://static.igem.org/mediawiki/2020/c/c7/T--SUSTech_Shenzhen--5-2-e-1024.png)](https://static.igem.org/mediawiki/2020/c/c7/T--SUSTech_Shenzhen--5-2-e-1024.png)

[![](https://static.igem.org/mediawiki/2020/7/70/T--SUSTech_Shenzhen--5-3-e-1024.png)](https://static.igem.org/mediawiki/2020/7/70/T--SUSTech_Shenzhen--5-3-e-1024.png)

Figure 4. Binding between Navitoclax and Aca1 confirmed using DSF

## Compound Design and Hit Optimization

In our previous work, we got a good result through a long period of time of virtual screening. Fortunately, the results of biological experiments show that the molecule has a certain regulatory effect on the protein *aca1*. However, the computer can only give us a rough calculation result based on the analysis of the volume of the protein pocket, and the molecule obtained does not show excellent performance in the interaction with the protein. Therefore, we need to analyze the molecular docking model, to optimize the structure of the hit, in order to improve its biological activity.

[![](https://static.igem.org/mediawiki/2020/c/cc/T--SUSTech_Shenzhen--6-1.png)](https://static.igem.org/mediawiki/2020/c/cc/T--SUSTech_Shenzhen--6-1.png)

Figure 5. Computer virtual molecular docking model of Navitoclax (in black) in complex with the *aca1*. Dotted arrows indicate interactions, and the spheres denote the surrounding amino acids.

We have obtained a hit compound through virtual screening and the docking model of it with *aca1* protein is shown in Figure 5. Through the analysis of the model, we can divide the structure of Navitoclax into five regions according to the binding strength of the structural fragment with the residue amino acids and the spatial structure of the protein pocket. The five regions are shown in Figure 6, distinguished by different colors.

[![](https://static.igem.org/mediawiki/2020/8/8c/T--SUSTech_Shenzhen--7-1024.png)](https://static.igem.org/mediawiki/2020/8/8c/T--SUSTech_Shenzhen--7-1024.png)

Figure 6. Five regions of Navitoclax. Dotted lines indicate the inner surface of the aca1 protein, and the five regions are distinguished with different colors.

According to the model, we found that two regions in the molecular structure should be retained in the subsequent molecular design and optimization. One of them is the structural segments of sulfonamides in Navitoclax (region III) because it plays an important role and exhibits strong interactions with LysA7 and AsnA13. We have reason to believe that this fragment may be a necessary structure for Navitoclax to be active in *aca1* protein. The other is the structure of morpholine (region I), which places in the solvent region of the pocket. Based on our understanding of the solvent region, there should be a water-soluble functional group to improve the binding ability with protein and improve the water solubility of the molecule. Morpholine, a highly water-soluble functional group can do this job excellently. So, this kind of structure will be retained in the following design and optimization.

However, another solvent region is occupied by a fat-soluble aromatic ring (region V). Moreover, no amino acid residue can interact with the aromatic ring, which is an unnecessary fragment. What’s more, the site has brought us some difficulties in chemical synthesis, which is not cost-effective in terms of economic benefits. Considering various factors, we decided to delete the aromatic ring above region V and modify the group below. We found that there is an acidic amino acid residue AspB75 at the bottom of region V. If we add an alkaline ring to this part, we have reason to believe that there will be a strong interaction between them. The common basic structure includes a large number of aromatic nitrogen heterocycles, saturated nitrogen heterocycles, and simple amine groups. But finally, we decided to use imidazole and triazole because they have the advantages of easy synthesis, moderate alkalinity, suitable size, proper rigidity, and so on.

Similarly, there is also a meaningless fragment in Region II, the thioether, which increases the cost of its synthesis but shows no significant interaction between the residue amino acids. As a result, we can replace the sulfur atom with a simple carbon atom. Then, considering there is an exposed basic amino acid residue LysA27 closed to Region II, an acidic group is used to replace the aromatic ring below to generate interaction with it. Generally speaking, the structure of carboxylic acid is a good choice. However, to enhance the membrane permeability of molecules, we can use the principle of bioisosteres to replace the structure of carboxylic acid with the structure of tetrazole.

In addition to analyzing the binding effect, we can also optimize our molecular structure by analyzing the spatial structure of the protein pocket. The optimization of region IV is a good example. Through the analysis of the spatial structure, we know that the space occupied by Region IV is not too wide. Therefore, we can choose to replace the ring structure of the region with an ordinary carbon chain or replace it with smaller rings. If we replace it with an ordinary fatty chain, the resulting molecule is likely to lose its rigidity and thus fail to perform the expected interaction well. Considering the above factors, we try to replace the region with an alkyne structure and five-membered heterocycles. At the same time, we have to consider whether we can optimize region V from the combination ability. We found that there are an amino acid residue CysA71 and PheA67 above region four, so we can use a structure of β-unsaturated carbonyl structure in region four to produce an n → π\* type of interaction with CysA71 or π→ π\*type of interaction with PheA67.

Through the theoretical analysis of the molecular docking model, we designed the second generation of small molecules to regulate Aca1 protein, and their structure and docking results are as follows.

[![](https://static.igem.org/mediawiki/2020/7/77/T--SUSTech_Shenzhen--1-14-1024.png)](https://static.igem.org/mediawiki/2020/7/77/T--SUSTech_Shenzhen--1-14-1024.png)

Figure 7. Optimized Aca1 inhibitors based on Navitoclax

Our second-generation molecules, in the design of regions I and II, chose to retain the same length of carbon chain as the first generation (Navitoclax). However, in the subsequent retrosynthesis analysis, we found that if we need to synthesize such a structural fragment (regions I and II), we need to use a polarity reversal dipole, such as α-halocarbonyl compound or cyanogen ion. Because α-halocarbonyl compounds have some problems in selectivity, and cyanogen compounds are highly toxic substances, which are rarely used in industry, even in the laboratory, which makes our synthesis and industrial synthesis very difficult.

[![](https://static.igem.org/mediawiki/2020/9/9b/T--SUSTech_Shenzhen--2-1-1.png)](https://static.igem.org/mediawiki/2020/9/9b/T--SUSTech_Shenzhen--2-1-1.png)

[![](https://static.igem.org/mediawiki/2020/5/52/T--SUSTech_Shenzhen--2-2-1-1024.png)](https://static.igem.org/mediawiki/2020/5/52/T--SUSTech_Shenzhen--2-2-1-1024.png)

Figure 8. Retrosynthesis of one Aca1 inhibitor

Therefore, we further designed the third-generation molecule to regulate Aca1 protein. The third-generation molecules further enrich the functional groups of molecular structure, so as to maximize the binding strength of molecules and proteins, so as to reduce the amount of actual use and reduce the cost. At the same time, we hope to optimize the physical and chemical properties of the molecules, such as water solubility. What’s more, we abandoned the structure fragments that are difficult to synthesize in the second-generation molecules, hoping to reduce the difficulty of synthesis and production costs. And their structures are shown below:

[![](https://static.igem.org/mediawiki/2020/3/36/T--SUSTech_Shenzhen--3-1-2-1024.png)](https://static.igem.org/mediawiki/2020/3/36/T--SUSTech_Shenzhen--3-1-2-1024.png)

[![](https://static.igem.org/mediawiki/2020/b/b6/T--SUSTech_Shenzhen--3-2-2-1024.png)](https://static.igem.org/mediawiki/2020/b/b6/T--SUSTech_Shenzhen--3-2-2-1024.png)

[![](https://static.igem.org/mediawiki/2020/a/a9/T--SUSTech_Shenzhen--3-3-1-1024.png)](https://static.igem.org/mediawiki/2020/a/a9/T--SUSTech_Shenzhen--3-3-1-1024.png)

[![](https://static.igem.org/mediawiki/2020/1/11/T--SUSTech_Shenzhen--3-4-1-1024.png)](https://static.igem.org/mediawiki/2020/1/11/T--SUSTech_Shenzhen--3-4-1-1024.png)

[![](https://static.igem.org/mediawiki/2020/8/80/T--SUSTech_Shenzhen--3-5-1-1024.png)](https://static.igem.org/mediawiki/2020/8/80/T--SUSTech_Shenzhen--3-5-1-1024.png)

Figure 9. Aca1 inhibitors (third generation)

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