iGEM 2022 — Team sustech-shenzhen

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* Project
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* Wetlab
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* Drylab
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  + [Directed Evolution](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27directed-evolution%27%29%20%7D%7D)
  + [Fermentation](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27fermentation%27%29%20%7D%7D)
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* HP
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  + [Education](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27education%27%29%20%7D%7D)
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  + [Inclusivity](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27inclusivity%27%29%20%7D%7D)
* Entrepreneurship
  + [Overview](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27entrepreneurship%27%29%20%7D%7D)
  + [EPIC VCL workshops](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27vcl-workshops%27%29%20%7D%7D)
  + [Summer bootcamp](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27summer-bootcamp%27%29%20%7D%7D)
  + [EPIC demo day](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27demo-day%27%29%20%7D%7D)
* Team
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![About Image](https://static.igem.wiki/teams/4425/wiki/notebook.jpg)

## Notebook

## 2022 May

Set up the 2022 SUSTech\_Shenzhen iGEM team

Determined that the project is to produce Tyrian Purple in E.coli with a reversible glucose protection

Found PIs to guide and guarantee our TPE project

Applied for funding from School of Life Sciences, SUSTech

## 2022 Jun

Ordered DNA synthesis during preparation of final exams

Plasmid construction of expressing UGT, PtUGT and BGL

Experimental skills training for beginners

## 2022 Jul

## 7.20 - 7.22

Obtained FMO, PtUGT and BGL fragments by PCR
Double digested plasmid pET28a
Select the monoclone of transformed E.coli and send for sequencing

## 7.23

FMO and PtUGT were ligated by overlapping PCR

## 7.25 - 7.26

Gibson construction of pET28a-PtUGT-FMO and pET28a-BGL and transformed to E.coli with plate growing
Select the monoclone of transformed E.coli and send for sequencing

## 7.28

Plasmid extraction of pET28a-PtUGT-FMO and pET28a-BGL

## 2022 Aug

## 8.5 - 8.7

The plasmids Fre-SttH, TnaA-FL-FMO and DH5α strain with endogenous TnaA gene knockout were obtained and transformed into E.coli with plate growing
Select the monoclone of transformed E.coli and cell cryopreserving

## 8.10

Fre-SttH, TnaA-FL-FMO strain plate streaking

## 8.11

Select the monoclone of transformed E.coli and amplification

## 8.12

The bacterial solution is diluted at a fold of 100 and incubated at 37 °C on a shaker at 200 rpm until OD 600 reached 0.6

0.3 mM IPTG is added for induction overnight

## 8.13

Determine soluble and whole cell sample by SDS-PAGE
Fermentation with glucose, Trp and Br-trp at 30 °C

## 8.14

The tyrian purple and indigo product after centrifugation was dissolved by DMSO and purified by washing with a large amount of water

## 8.17

Dye fabric strips with tyrian purple and indigo

## 8.21

SfGFP fragment was obtained by PCR
Double digested plasmid pPOS and pNEG

## 8.22

Gibson construction of pNEG-sfGFP and pPOS-sfGFP
Transform two plasmids in E.coli with plate growing

## 8.23

Select the monoclone of transformed E.coli and send for sequencing

## 8.25

Plasmid extraction of pNEG-sfGFP and pPOS-sfGFP
Prepare pNEG and pPOS competent cells

## 2022 Sep

## 9.3

Prepare 2xYT medium, pH adjustment and sterilization

## 9.6

pTrpR was transferred into pNEG competent cells

## 9.7

Went through GFP test. Different concentrations of IPTG were added for induction with adding the substrate Br-Trp, and the fluorescence intensity was detected by microplate reader

## 9.13

The emulsion was configured and mixed with the cell suspension induced to produce sfGFP, and the quality of emulsion was detected by phase contrast microscopy

## 9.20

INAC-pTrpR was constructed by inserting random sequences into pTrpR

## 9.21

INAC-pTrpR and pTrpR were mixed into 1:1, 1:10, 1:100 and 1:1000, and SDS-PAGE was performed after PCR

## 9.23

INAC-pTrpR and pTrpR were mixed at the ratio of 1:1 and converted into pNEG competent cell.
CPR cycle was performed, and the product was collected to run agarose electrophoresis

## 9.24

The colonies on the plate were scraped to buid up formal library
Plasmid extraction to obtain No.0 library

## 9.27

No.0 library was transformed into competent cell transformed with pNEG

## 9.28

CPR cycle was performed, and product was cleaned and concentrated using a PCR purification column after run a agarose electrophoresis

## 9.29

Assemble DNA product in 0928 with pTrpR backbone to get No.1 library.

## Future Plan

## After wiki freeze but before Grand Jamboree:

Check the size and analyze the selection result of No.1 library

Transform pET28a-PtUGT-FMO and pET28a-BGL into BL21 competent cell and try to produce indican

Supplement quantitative experiments to fermentation of producing tyrian purple by LC-MS

## After iGEM:

Continue directed selection of No.1 library, and check the size and analyze the selection result of library in each cycle

Find other residue combinations, and construct new library for selection

Use the muted trp repressor to construct sensor system to test promotor expression in different ratio of trp and br-trp

#### Notebook

* [2022 May](#2022 May)
* [2022 June](#2022 Jun)
* [2022 Jul](#2022 Jul)
* [2022 Aug](#2022 Aug)
* [2022 Sep](#2022 Sep)
* [Future Plan](#Future Plan)

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© 2022 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
The repository used to create this website is available at <https://gitlab.igem.org/2022/sustech-shenzhen>.

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* Drylab
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  + [Directed Evolution](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27directed-evolution%27%29%20%7D%7D)
  + [Fermentation](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27fermentation%27%29%20%7D%7D)
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* HP
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  + [Integrated Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27human-practices%27%29%20%7D%7D)
  + [Education](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27education%27%29%20%7D%7D)
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  + [Inclusivity](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27inclusivity%27%29%20%7D%7D)
* Entrepreneurship
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  + [EPIC VCL workshops](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27vcl-workshops%27%29%20%7D%7D)
  + [Summer bootcamp](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27summer-bootcamp%27%29%20%7D%7D)
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* Team
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![About Image](https://static.igem.wiki/teams/4425/wiki/engineering.jpeg)

## Engineering

## Overview

Engineer and optimize TPE project is our core task for wetlab. Just like the strategy of directed evolution, we believe that an interesting project needs to be “evoluted”. We followed a “Design → Build → Test → Learn” loop to achieve the “evolution” of TPE project. There are two parts of optimization in the project:

We use directed evolution to develop the process of synthesis from two-cell system to one-cell system, with an expected optimization controlling system of endogenous TnaA enzyme in E.coli.

We introduce a reversible glucose protection group to make the production of tyrian purple dye more stable and convenient for industrial scale-up.

Thus, we did the “Design → Build → Test → Learn” loop for each of optimization, making them become highlights in TPE project.

## Establishing a pathway from Trp to 6-bromoindole

### Design

SttH is a trp-6-halogenase that requires FADH2 as a co-factor to convert tryptophan (Trp) into 6-Br-trp. Tryptophanase TnaA can cleave Trp into indole or cleave 6-Br-Trp into 6-Br-indole. Thus, 6-Br-indole can be synthesized via two routes using SttH and TnaA: Previous conversion of Trp to indole followed by bromination of indole or bromination of Trp followed by conversion to 6-Br-indole.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering1.png)

##### FIGURE 1

Two possible pathways from Trp to 6-bromoindole

### Build

To validate the two pathways, all the enzymatic steps using TnaA or SttH were individually evaluated through whole-cell reactions. To prevent the effect of endogenous TnaA expression, a ΔtnaA strain of E. coli was used and TnaA was overexpressed using a plasmid when needed.

Thanks to 2021 iGEM team LINKS\_China for providing us with their parts:
part BBa\_K4011004 (TnaA-FL-FMO) and part BBa\_K4011003 (Fre-SttH)

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering2.png)
![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering3.png)

We used plasmids offered by LINKS\_China to cut and ligation for obtaining plasmids expressing SttH and TnaA.

### Test

Firstly, we found that SttH alone had low reactivity because of mostly existing in an insoluble form[1]. Thus, we interspersed another ENGINEER process at the same time to solve this problem which is shown on the next part of engineer webpage.
With literature survey and review, the test result shows that when 2.5 mM indole or Trp was used as substrate, 0.29 mM of 6-Br-indole or 0.78 mM of 6-Br-Trp was produced (Figure 2).

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering4.png)

##### FIGURE 2

Concentrations of the substrates (indole or Trp) and the products (6-Br-indole or 6-Br-Trp) after the whole-cell reactions of ΔtnaA SttH+Fre using indole or Trp, respectively.

### Learn

Overall, the activity data suggest that the SttH reaction followed by the TnaA reaction (Trp→6-Br-Trp→6-Br-indole) give a higher yield of 6-Br-indole and would be advantageous for 6BrIG production over the other route (Trp→indole→6-Br-indole).

## Construction of Trp halogenase (SttH) and Fre fusion enzyme

### Design

We found that SttH alone had low reactivity because of mostly existing in an insoluble form, possibly limiting the conversion of bromination up to 31.2% (0.78mM 6-Br-Trp, Figure 2)[1]. To improve the bromination reaction rate, we followed the designing of Lee et al to increase the solubility of SttH by constructing a fusion enzyme with a highly soluble protein at the N terminus of SttH (Figure 3). Fre was chosen as a fusion partner for SttH, since it is highly expressed in a soluble form in E. coli and can regenerate FADH2 efficiently.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering5.png)

##### FIGURE 3

SttH and Fre fusion enzyme with a flexible linker

### Build

Thanks to 2021 iGEM team LINKS\_China for providing us with their parts:
part BBa\_K4011003 (Fre-SttH)

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering6.png)

### Test

Although the total expression of SttH decreased, the chimeric enzyme showed highly increased ratios of their soluble forms in E. coli with a higher field of 6-Br-trp. The molar concentrations of soluble Fre-L–SttH was 1.64 μM, which was more than three times higher than that of SttH by itself (0.53 μM)[1].

### Learn

In summary, Fre was effective as a soluble N-terminal tag for SttH. And we choose Fre-SttH to replace alone SttH for Trp bromination in the last engineering process.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering7.png)

##### FIGURE 4

Reaction scheme of Trp bromination.
Trp 6-Hal requires oxygen, bromide salt and FADH2.
Flavin reductase (Fre) regenerates FAD to FADH2 using NAD(P)H.

## Fermentation of Tyrian purple in two-cell system of E. coli

### Design

The reported scheme of tyrian purple production in E.coli with the substrate of Trp is shown in Figure 5. We designed our optimization based on this scheme.
As natural dye indigo and tyrian purple is hard to solute in water and cause difficulty in cloth dying. We applied the strategy that introduce a glucose moiety as a reversible protecting group to the reactive indigo and tyrian purple precursor indoxyl by glucosyltransferase PtUGT to form soluble indican as fermentation product (Figure 5). And β-glucosidase BGL is applied to remove the protecting group when cloth dying[2].

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering8.png)

##### FIGURE 5

Reaction schemes of Trp 6-halogenase (Trp 6-Hal), tryptophanase (TnaA) and monooxygenase (MO)[1]

Also, for the steps using TnaA and MO (using one of special kinds named flavin-containing monooxygenase, MaFMO or FMO), we linked them with a flexible linker to form a chimeric enzyme for higher field and efficiency.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering9.png)

##### FIGURE 6

Optimized reaction scheme with the reversible protection of glucose

### Build

Thanks to 2021 iGEM team LINKS\_China for providing us with their parts:
part BBa\_K4011004 (TnaA-FL-FMO) and part BBa\_K4011003 (Fre-SttH)

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering10.png)
![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering11.png)

We constructed plasmid pET28a-FMO-PtUGT to co-express PtUGT and FMO to convert indole or 6-Br-indole to indican or 6-Br-indican respectively. We also construct plasmid pET28a-BGL and pET28a-FMO to individually express BGL and FMO.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering12.png)
![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering13.png)
![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering14.png)

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering15.png)

##### FIGURE 7

Construction of pET28a-FMO-PtUGT

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering16.png)

##### FIGURE 8

Construction of pET28a-FMO

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering17.png)

##### FIGURE 9

Construction of pET28a-BGL

### Test

We transformed parts TnaA-FL-FMO and Fre-sttH into DH5α E.coli which endogenous TnaA gene have been knocked out. After culture and induced expression of these strains, we use the SDS-PAGE to separate and show the expression of TnaA-FL-FMO and Fre-sttH (Figure 10A). We found that we successfully express our fused proteins and these two proteins show high water solubility.

We then culture strain that has been induced to express Fre-sttH by NPB solution and 2.5mM trp and 300mM NaBr as substrate. During fermentation of 24h, we use LC-MS to obtained data of concentration of trp and 6-Br-trp by every 6h (Figure 10B). Strain that has been induced to express TnaA-FL-FMO also be cultured by NPB solution and 1mM trp or 6-Br-trp as substrate. After fermentationfor 24h, we get obvious blue and purple dye in our culture tubes (Figure 10C). Besides, we attempt to produce tyrian purple by TnaA-FL-FMO using the culture after fermentation of trp and NaBr with Fre-sttH and the result has no difference from fermentation using standard 1mM 6-Br-trp (Figure 10D).

Considering the bad solubility of indigo and tyrian purple, we apply 100% DMSO to solute the dye from cell precipitation and we get DMSO solution of indigo and tyrian purple (Figure 10E). Dye concentration is calculated by a standard curve measured by LINKS\_China. We get a ~30% yield for indigo and ~20% yield for tyrian purple.
We also use the tyrian purple we produce in cloth dying by put a piece of cloth into fermentation culture of Br-trp with TnaA-FL-FMO and we finally get the cloth in purple (Figure 10F).

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering18.png)

##### FIGURE 10A

 SDS-PAGE of TnaA-FL-FMO and Fre-sttH. WC stand for whole cell sample and S stand for supernatant sample after cell lysis

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering19.png)

##### FIGURE 10BCDEF

 B) Concentration-time graph of trp and 6-Br-trp in culture of 2.5mM trp and 300mM NaBr with Fre-sttH. C) Tyrian purple (left) and indigo (right) produced by 6-Br-trp and trp with TnaA-FL-FMO. D) Tyrian purple produced by using culture of fermentation with Fre-sttH and trp. E) DMSO solution of indigo (top) and tyrian purple (bottom). F) Frabic dyed with tyrian purple.

### Learn

Our design of optimized reaction scheme is smooth and successful with a high yield of precursors indican, Br-indican and final product indigo and tyrian purple, as well as the excellent ability to dye fabrics evenly. Also, we can draw the conclusion that the constructed parts all work well and perform their correct function.

## Optimized fermentation of Tyrian purple in one-cell system of E. coli

### Design

Due to TnaA can react with both tryptophan and the first step product bromotryptophan, Fre-SttH and TnaA has to be in two different strains. So the two-cell system, ΔTnaA E. coli strain without endogenous TnaA expression and another ΔTnaA E. coli strain with extra TnaA expression, are used to produce tyrian purple with less byproduct indigo. That’s why Lee et al have to use two-cell system to complete the production of tyrian purple.

Considering the problem of tryptophanase TnaA lack of specificity, we want to use muted trp repressor that can only bind trp to sense the concentration of trp and 6-Br-trp in E. coli to control the expression of TnaA. We optimized two-cell sysytem to one-cell system with a spatio-temporal well-controlled TnaA synthesis (Figure 11). We applied a synthetic phylogeny of programmable Trp repressor with directed evolution to E.coli, which simplified the process of reacting with bromotryptophan not tryptophan[3].

Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype–genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection[4].

Iterative rounds of positive and negative compartmentalized partnered replication (CPR) led to the exponential amplification of variants that responded with high affinity and specificity to halogenated tryptophan analogs and novel operator sites. We empower cells to perceive the relative ratio of bromotryptophan to tryptophan to control TnaA synthesis.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering20.png)

##### FIGURE 11

Optimized reaaction scheme of one-cell system adding directed evolution

### Build

We firstly constructed three plasmids pTrpR, pPOS and pNEG. pTrpR is induced to express muted trp repressors. Active trp repressors with its ligand can induce pPOS to express Taq polymerase and inactive trp repressors or trp repressor without its ligand can induce pNEG to express Taq polymerase (Figure 12).

For GFP test of CPR cycle, we constructed pNEG-sfGFP and pPOS-sfGFP which replace Taq polymerase gene with sfGFP to characterize the expression of Taq polymerase.

For the mock selection of CPR cycle, we inserted a DNA sequence of 200bp into wild type trp repressor to construct INAC-pTrpR (Figure 18). We mix plasmid pTrpR with wild type trp repressor and INAC-pTrpR with a mass ratio of 1:1 and transform the mixture into pNEG and pPOS competent cells. Induced cells were resuspend by CPR mix and emulsion droplets were constructed by mixing CPR mix and oil mix and vortex for 4 min.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering21.png)

##### FIGURE 12

Scheme of CPR positive and negative circuits

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering22.png)

##### FIGURE 13

Construction of pTrpR

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering23.png)

##### FIGURE 14

Construction of pPOS

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering24.png)

##### FIGURE 15

 Construction of pNEG

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering25.png)

##### FIGURE 16

 Construction of pPOS-GFP

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering26.png)

##### FIGURE 17

Construction of pNEG-GFP

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering27.png)

##### FIGURE 18

Scheme of INAC-pTrpR construction

### Test

(1) GFP test

The expression of Taq polymerase can not be monitored easily, we apply GFP assay to use GFP production to analyzing the gene circuits function. We transform pTrpR which express wild type trp repressor into competent cells that have been transformed with plasmid pNEG-sfGFP or pPOS-sfGFP. Then we culture and induce with different concentration of trp and 1mM IPTG to induce expression of sfGFP. We expect that as trp concentration increases, the expression of sfGFP (RPU/OD600) in pNEG-sfGFP gradually decreases, and the expression of sfGFP (RPU/OD600) in pPOS-sfGFP gradually increases (Figure 19A).

(2) Mock selection

In addition to GFP test, we also perform a mock selection to confirm the designed gene circuit function can actually select positive clone. At first, we mix culture with strains that has been induced to express sfGFP and oil mix to check the quality of emulsion droplet. We measure the diameter of droplet with a phase contrast microscopy. We found that the diameter of emulsion droplets are range about 2-10 μm and less than 20% emulsion droplets contain a GFP-expression cell (Figure 19B). Thus, we have checked any emulsion droplet can only contain a single cell. We insert a random sequence into wild type trp repressor to make it inactive to bind to trp, and we get plasmid INAC-pTrpR. We mix plasmid pTrpR with wild type trp repressor and INAC-pTrpR with a mass ratio of 1:1 and transform the mixture into pNEG and pPOS competent cells. A mock CPR seletion is next carried out. Induced strains is emulsified with emulsion mix and replicate positive clone by ePCR. The emulsion remains in a phase-independent state during ePCR (Figure 19C). After breaking the emulsion by adding ddH2O and chloroform, we apply a recovery PCR and perform a agarose electrophoresis to monitor the result of the mock selection. PNEG give an obvious amplification of inactive trp repressor and pPOS give an obvious amplification of active trp repressor (Figure 19D).

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering28.png)

##### FIGURE 19A

GFP production (RPU/OD600) – trp concentration graph by GFP test for wild type trp repressor and pNEG (due to time and pandemic, we only completed the experiment of pNEG).

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering29.png)

##### FIGURE 19B

 Phase contrast microscopy image of emulsion droplet of 50×diluted scale bar, 10μm (left) of emusion droplet merge with GFP expression of 1× diluted emulsion scar bar, 10μm.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering30.png)

##### FIGURE 19C

From left to right: cell culture emulsified with emulsion mix; Cell emulsion after ePCR circle; Emulsion after perform centrifuge; Phase-separate solution after breaking emulsion.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering31.png)

##### FIGURE 19D

 result of agarose electrophoresis between trpR:INAC-trpR = 1:1 and trpR and inactive trpR after mock selection with pNEG (due to time and pandemic, we only completed the experiment of pNEG)

### Learn

We found that high concentration of trp can inhibit expression of sfGFP in negative selection system. However, it can only provide about three times the inhibition effect which cannot achevie screen effect. Possibly, trp repressor has a low expression or trp repressor with trp cannot strongly bind to trp operator at DNA. Same experiments of positive selection system haven't be carry out.

After a literature survey, we found that expressing GFP with an enhanced Trp+ promoter may be a good solution.

Also, we have successfully make emulsion droplets of high quality. However, results of mock selection is not obvious which means selection system cannot distinguish wild type trp repressor with inactive trp repressor that consistent with results of GFP test.

## Directed evolution of trp repressor mutation library

### Design

After testing the CPR circuit works well, we are going to introduce directed evolution to trp repressor. Our libraries are designed in which residues (I57, V58, R54, L60, G85, T81, I82) that might contact trp at its 6C positions of the indole ring due to their proximity. Considering several position overlapped with those involved in the formation of the HTH motif, we initially choose I57, V58 and T81 as our mutant site for directed evolution. We perform site-directed saturation mutation on these three residues by completely randomized through re-synthesis of the gene with NNS codons (N = A, T, G, C; S = G, C). The library (32768 variants) are transformed into competent cells that have been transformed with pNEG or pPOS beforehand and carry out several circle of CPR to select positive clone we want (Figure 20). Due to the restriction of time, we have not perform a selective circle in our project, we plan to simulate this process using mathematical modeling.

Also, for the steps using TnaA and MO (using one of special kinds named flavin-containing monooxygenase, MaFMO or FMO), we linked them with a flexible linker to form a chimeric enzyme for higher field and efficiency.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering32.png)

##### FIGURE 20

3D view of trpR binding to DNA and the trp binding site

### Build

More details in [Drylab](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27model%27%29%20%7D%7D).

### Test

At first, we modeling the process of Transcription and translation process. We used the ODE solver “deSolve” in R to simulate our positive selection system based off the 9 ODEs we wrote and “ggplot2” to plot the dynamics of molecules of interest, and obtained the results below: As shown by the graph (Figure 21A), within 2 hours, TrpR reaches its plateau at about 150 uM and at the same time TrpR gets rapid polymerization formation into TrpR2, which peaks at Time = 20h, 75 uM. Compared with TrpR and TrpR2, TrpR2-T's formation process is much lower, reaching it saturated at Time = 40h, 25 uM. In the CI, CI2 graph (Figure 21B), CI quickly reaches its peak at Time = 8h, 3.8 uM and then it go through a steady but slow decreasing process to about 1.7 uM. While for the CI2 protein, it has the similar trend with CI and its peak and steady state concentration is half of that of CI. While for the GFP graph (Figure 21C), we could see in the first day (within 24h), GFP quickly peaks at about 2 uM, while after that it experiences a significant drop to no more than 0.5 uM. Soon, GFP quickly get translation.

Then, we model the influence of Trp's concentration in directed evolution system X aix means Trp increase from 10-4 to 1, Y aix means relatively expression level (Figure 21D). In the left figure, we can see, when Trp's concentration is under 20 × 10-3 uM. CI2 protein is at its saturate state. When Trp increases to 30 × 10-1 uM, CI2 experiences a significant drop, from 2.0 uM to 0.5 uM. In the right figure, GFP's relative expression process is contrary to CI2 trend. Below Trp concentration 20 × 10-3 uM, GFP's expression is close to 0. But as trp's concentration increases.

![Blog One](https://static.igem.wiki/teams/4425/wiki/engineering33.png)

##### FIGURE 21

 A) TrpR, TrpR2 and TrpR2-T expression changed with time B) CI and CI2 expression changed with time C) GFP expression changed with time D) CI2 and GFP expression changed with increased Trp concentration.

### Learn

With the help of modeling, we know better about this directional evolution and selection system. As seen in the results, our modeling shows how the positive screening system works. TrpR and TrpR2 quickly reach its saturate state with proper Trp concentration, and TrpR2 and Trp's polymer can generate, which can inhibit the expression of CI2. Also, Taq enzyme can be expressed. With increasing Trp concentration, the relative expression of GFP has a similar trend with experiment data in wetlab. According to this, we could estimate the combined rate between TrpR2 and Trp, which has a huge benefit to our gene design.

## Summary

Our project optimized the synthesis biology approach to product tyrian purple and verified feasibility of synthesizing tyrian purple with a one-cell system. Mathematical modeling results can be used as a reference of quantitative data in wetlab.

## References

[1] Lee, J., Kim, J., Song, J. E., Song, W. S., Kim, E. J., Kim, Y. G., Jeong, H. J., Kim, H. R., Choi, K. Y., & Kim, B. G. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nature chemical biology, 2021, 17(1), 104-112.
DOI: https://doi.org/10.1038/s41589-020-00684-4
[2] Hsu, T. M., Welner, D. H., Russ, Z. N., Cervantes, B., Prathuri, R. L., Adams, P. D., & Dueber, J. E. Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nature chemical biology, 2018, 14(3), 256–261.
DOI: https://doi.org/10.1038/nchembio.2552
[3] Ellefson, J. W., Ledbetter, M. P., & Ellington, A. D. Directed evolution of a synthetic phylogeny of programmable Trp repressors. Nature chemical biology, 2018, 14(4), 361–367.
DOI: https://doi.org/10.1038/s41589-018-0006-7
[4] Abil, Z., Ellefson, J. W., Gollihar, J. D., Watkins, E., & Ellington, A. D. Compartmentalized partnered replication for the directed evolution of genetic parts and circuits. Nature protocols, 2017, 12(12), 2493–2512.
DOI: https://doi.org/10.1038/nprot.2017.119

#### Engineering

* [Overview](#Overview)
* [Establishing a pathway from Trp to 6-bromoindole](#Establishing a pathway from Trp to 6-bromoindole)
* [Construction of Trp halogenase (SttH) and Fre fusion enzyme](#Construction of Trp halogenase (SttH) and Fre fusion enzyme)
* [Fermentation of Tyrian purple in two-cell system of E. coli](#Fermentation of Tyrian purple in two-cell system of E. coli )
* [Optimized fermentation of Tyrian purple in one-cell system of E. coli](#Optimized fermentation of Tyrian purple in one-cell system of E. coli)
* [Directed evolution of trp repressor mutation library](#Directed evolution of trp repressor mutation library)
* [Summary](#Summary)
* [References](#References)

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## Experiments

![Blog One](https://static.igem.wiki/teams/4425/wiki/experiment0.png)

##### FIGURE 1

Outline of experiments

## Prodecu Tyrian Purple and indigo with two-cells system

### Reagents

Plasmid; Resistant medium with antibiotics; HEPES solution; NaCl solution; Imidazole solution; Protein buffer; Chloroform; Ventilation cabinet; Ethyl acetate; Tryptophan; 6-chloro-tryptophan; 6-bromotryptophan, Methanol.

### Equipments

Centrifuge tube; 37 ℃ shaker; OD measurement instrument; Centrifuge; Ultrasonic instrument; Filter screen.

### Procedure

(1)Expression and reaction of TnaA-FMO

Single colonies are selected and incubated in LB medium with antibiotics overnight. The next day, the bacterial solution is diluted 1:100 and incubated in a 37 °C and 200 rpm shaker until the OD 600 value reached 0.6. After that, 0.3 mM IPTG is added for overnight induction. Next, the bacterial solution is centrifuged at 3,500 × g for 10 min, the excess LB is washed with PBS, and the cells are resuspended with NPB solution of 50 mM and pH=8.0. Final reaction system is: total volume 4 mL,OD 600=20,Trp (BR-TRP) 1 mM, glucose 0.6%. The reaction is performed at 30 °C for 24 h.

After the reaction, the bacterial solution is partitioned into a 1.5 mL centrifuge tube, centrifuged at 13,000 × g for 10 min, and indigo is dissolved with 50% DMSO in H2O or 100% DMSO.

(2)Expression and reaction of Fre-SttH

Single colonies are selected and incubated overnight in LB medium with antibiotics. The next day, the bacterial solution is diluted 1:100 and incubated in a 37 °C and 200 rpm shaker until the OD 600 value reached 0.6. Then 0.3 mM IPTG is added for overnight induction. Next, the bacterial solution is centrifuged at 3,500 × g for 10 min, the excess LB is washed off with PBS, and the cells are resuspended with NPB solution with a concentration of 50 mM and pH=8.0. The final reaction system is as follows: The total volume is 4 mL, OD 600=20, Trp 2.5 mM, glucose 0.6%, NaBr 300 mM, 30 °C. Reaction for 24h.

(3)Preparation of SDS-PAGE samples

The induced bacterial solution is centrifuged at 4,000 rpm for 10 min and resuspended in PBS. Then 15 uL of whole cell sample is taken. The remaining bacterial solution is lysed by ultrasonic wave and centrifuged at 13,000 × g for 2 min. Next, 15 uL of supernatant sample is taken. After mixing with sample buffer for protein, the sample is boiled at 100°C for 5min before loading.

(4)LC-MS sample processing

 **Br-trp, Trp:**
1 mL of reaction solution is centrifuged at 13,000 × g for 10 min, and the supernatant is filtered through a 0.22 um filter.

 **Indole, Br-indole:**
200 uL of reaction solution is added with 400 uL CHCl3 and removed 200 uL CHCl3 after vortex. The mixture is evaporated in a fume hood and the remainder dissolved in 100 uL of ethyl acetate.

**Indican:**
The reaction solution is filtered through a 0.22 um filter and diluted with 10 times water.

(5)LC-MS detection conditions

**Samples to be tested:** tryptophan, 6-chloro-tryptophan, 6-bromotryptophan
**Chromatographic Column:** C18 chromatographic column
 **Mobile phase:**
A: 0.3%TFA/ water
B: 0.3%TFA/ methanol
**Speed:** 1 mL/min
**etection wavelength:** 280 nm

![Blog One](https://static.igem.wiki/teams/4425/wiki/experiment5.png)

(6)Fabric dyeing with Br-indican

Indican median is dribbled onto cotton cloth to moisten, and then dried by adding 500 uL of BGL lysate to allow spontaneous oxidation.

(7)Fabric dyeing with tyrian purple

Dyeing solutions (DI water 50 mL; tetrahydrofuran 7.5 mL; NaOH 0.5 g; Na2S2O4 0.5 g and chemical 6BrIG 0.15 g or the produced 6BrIG cell dye) are prepared by heating with stirring on the 75-80 °C hot plate for 15 min. After heating, 0.5 g NH4Cl is added to the dyeing solution and subsequent heating is repeated at 75-80 °C. The fabrics (multi-fabric strips or wool fabrics) are soaked into the solution and dye at 60 °C for 15 min.

## Introduce a reversible protecting glucose group onto indoxyl to produce indican

### Reagents

Plasmid; Resistant medium with antibiotics; HEPES solution; NaCl solution; Imidazole solution; Protein buffer; Chloroform; Ventilation cabinet; Ethyl acetate; Tryptophan; 6-chloro-tryptophan; 6-bromotryptophan, Methanol.

### Equipments

Centrifuge tube; 37 ℃ shaker; OD measurement instrument; centrifuge; Ultrasonic instrument; Filter screen.

### Procedure

(1)Expression, purification and reaction of UGT-FMO

The plasmid PET28A-FMO-ptUGT is transformed into strain DE3 and cultured overnight in LB medium with resistance. The next day, the bacterial solution is diluted at a fold of 100 and incubated at 37 °C on a shaker at 200 rpm until OD 600 reached 0.6. After that, 0.3 mM IPTG is added for induction overnight (the induced bacterial solution can be resuspended by buffer solution of HEPES with a concentration of 50 mM and pH 7.0, sodium chloride solution with a concentration of 300 mM, imidazole solution with a concentration of 25 mM and pH 8.0, and DTT solution with a concentration of mM.) Finally, UGT is purified by cleavage.

(2)Reaction detection

After incubation overnight to saturation, the bacteria solution is diluted in 50 mL M9 medium until OD 600 reached 0.05. Then 26 mM Trp, 2% glycerol, 0.2% glucose, 1 mM IPTG are added and the reaction is performed at 37 °C and shaked at 200 rpm. During this time, the content of bacteria and reactants should be measured regularly.

## GFP Test and mock selection to test the effectiveness of CPR circle

### Equipments

**GFP test**

96-Well black microplates (Corning, cat. no. CLS3915); 96-Well clear microplates (Corning, cat. no. 3370); Microplate reader (Tecan, M200 PRO).

**Emulsion PCR**

Spectrophotometer 1 mL Syringes; TissueLyser; Microman pipette for viscous liquids; Capillary pistons; Inverted Epi-Fluorescence Phase Contrast Microscope; FITC/GFP filter cube; Cellometer cell counting chambers; Microscope camera calibration slide; ImageJ.

### Procedure

Centrifuge tube; 37 ℃ shaker; OD measurement instrument; centrifuge; Ultrasonic instrument; Filter screen.

### Procedure

(1)GFP Test
1.Tryptophan analog responsive variants, 5R and 6R, are transformed into JW4356 E. coli cells.
2.Depending on direct or inverted circuit screening architecture, these are co-transformed with either pNEG+GFP or pPOS+GFP for an inverted signal.
3.Overnight cultures of individual colonies are seeded into a 2 mL 96-well grow block containing 2xYT supplemented with ampicillin, chloramphenicol, and 1 mM IPTG at a 1:20 dilution ratio.
4.Wells contained the indicated concentration of either 5-bromo-L-tryptophan (Sigma) or 6-bromo-L-tryptophan (Gold Biotechnology).
5.Cells are incubated for 5 h at 37 % CO2 to allow expression of repressors and GFP.
6.Prior to fluorescence measurement on a plate reader (TecanM 200, excitation 469 nm; emission 501 nm), cells are centrifuged at 4 °C for 20 min at 3,000 × g and resuspended in 1X PBS solution.
7.Fluorescence intensities are measured and normalized to the OD 600 of the culture. Background JW4356 cell fluorescence is subtracted from all samples, which is calculated by the fluorescence divided by OD 600 for the parental JW4356 strain.
8.Maximal signal output is calculated by transformation of a control vector with wild-type TrpR (for inverter circuit) or inactivated TrpR (for repression circuit).
9.To calculate the half maximal effective concentration (ECs) for repression, the dose-response function for either 5R or 6R is fit to the equation:
![Blog One](https://static.igem.wiki/teams/4425/wiki/experiment6.png)

where c is the EC50 of 5- or 6-bromo-L-tryptophan (mM),
A is the maximum response,
x is the ligand concentration,
b is the hill coefficient.

(2)Mock selection
1.Both selection plasmids (pPOS and pNEG) are functionally tested by expression of GFP under active or repressed conditions.
2.To test: PLacUV5 drove expression of either a functional TrpR or an inactive TrpR (truncated by two stop codons (K27TAG and Y30TAA) and containing a unique internal HindIII restriction site; plasmid pTrpR.2TAA).
3.To test positive or negative selection CPR, these control constructs are co-transformed with the Taq expressing pPOS or pNEG vectors.
4.A single round of CPR selection is performed (detailed below) for both positive and negative selections using various ratios of active or inactive repressor (1:10 to 1:10,000). Under these conditions, positive selection should enrich for active TrpR repression, whereas negative selection should enrich for inactive TrpR repressors.
5.The enrichment factor for positive selection is estimated to be roughly 300-fold per round of CPR, while the negative selection is estimated to be roughly 200-fold per round of CPR. These are calculated using the following formula:
![Blog One](https://static.igem.wiki/teams/4425/wiki/experiment7.png)

where (X initial/Y initial) is the initial ratio of active or inactive repressor, EF is the enrichment factor, and (X final/Y final) is the ratio of active or inactive repressor post CPR selection.

## Carry out several CPR circles to select the trp repressor mutant that can only bind to trp

### Reagents

Tris base; Hydrochloric acid; Sodium chloride; Tegosoft DEC (diethylhexyl carbonate; Evonik, cat. no. 14858–73–2); Mineral oil; Abil WE 09 (Evonik); Potassium chloride; Magnesium chloride; dNTP mix, 10 mM each; PCR primers; Tetramethyl ammonium chloride (TMAC) solution, 5 M RNase A, 1 mg/ml; Chloroform; 5Prime Phase Lock Gel Heavy tubes; Restriction enzyme Dpnl
CutSmart Buffer, 10× QIAquick PCR Purification Kit; Sodium acetate; Glacial acetic acid.

### Equipments

Spectrophotometer 1 mL Syringes; TissueLyser; Microman pipette for viscous liquids; Capillary pistons.

### Procedure

1.TrpR repressor libraries are created through site-saturation mutagenesis using degenerate oligonucleotides with NNSor NDS codon randomization.
2.Libraries are clonedinto an ampicillin-resistant ColE1 origin plasmid containing the IPTG inducible lacUV5 promoter. Libraries are electroporated into E. coli strain JW4356
(a tryptophan repressor knockout strain) pre-transformed with either the positiveor negative selection vector.
3.Initial library sizes are above the theoretical diversitythreshold (~ 3.0 × 107) and are maintained throughout the CPR selection, witha transformation efficiency of at least 106, but more typically 107-108. Overnightlibrary cultures are diluted 1:20 into fresh 2xYT media supplemented with 100 ug/mL ampicillin, 34 ug/mL chloramphenicol, and 1 mM of 5-bromo-L-tryptophan (Sigma- Aldrich, 99% purity) or 6-bromo-L-tryptophan (Gold
Biotechnology, 99.7% purity), when necessary. Cells are grown for 1 h at 37 °C and subsequently induced by the addition of 1 mM IPTG and incubated at 37 °C for an additional 4 h.

 4.Induced cells (200 uL total) are spun in a tabletop centrifuge at 3,000 × g for 8 min. The supernatant is discarded, and the cell pellet is resuspended in 150 uL CPR mix: 1x Taq buffer (10 mM Tris-HCl with pH 8.3, 50 mM KCl, 1.5 mM MgSO4), 260uM dNTPs, 530 nM CPR selection primers (JE.TrpR.CPR.F and JE.TrpR.CPR.R).

 5.The resuspended cells are placed into a 2 mL tube with a 1 mL rubbersyringe plunger and 600 uL of oil mix (73% Tegosoft DEC, 7% AbilWE09 (Evonik), and 20% mineral oil (Sigma-Aldrich)).

 6.The emulsion is created by placing thecell and oil mix on a TissueLyser LT (Qiagen) with a program of 42 Hz for 4 min.The emulsified cells are thermal-cycled with the program: 95 °C for 3 min, 25 cycles (95 °C for 30 s, 55% CO2 for 30 s, 72 °C for 1 min).

 7.Emulsions are broken by spinningthe reaction (10,000 × g for 5 min), removing the top oil phase, adding 150 uL of H2O and 750 uL chloroform, vortexing vigorously, and finally, phase separating in aphase lock tube.

 8.DNA in the aqueous phase is cleaned and concentratedusing a PCR purification column. CPR-amplified TrpR genes are isolated through PCR using outnested recovery primers (JE.TrpR.Recov.F and JE.TrpR.Recov.R). Typically, this is achieved by addition of 1/10 the total purified DNA using.

 9. Accuprime Pfx (Thermo Fisher) in a 20-cycle PCR; however, challenging rounds ofselection could require increasing the amount of template DNA or cycle number toachieve detectable amplification. Resulting DNA products are re-cloned into theselection vector, completing one full round of CPR selection.

#### Experiments

* [Prodecu Tyrian Purple and indigo with two-cells system](#Prodecu Tyrian Purple and indigo with two-cells system)
* [Introduce a reversible protecting glucose group onto indoxyl to produce indican](#Introduce a reversible protecting glucose group onto indoxyl to produce indican)
* [GFP Test and mock selection to test the effectiveness of CPR circle](#GFP Test and mock selection to test the effectiveness of CPR circle)
* [Carry out several CPR circles to select the trp repressor mutant that can only bind to trp](#Carry out several CPR circles to select the trp repressor mutant that can only bind to trp)

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The repository used to create this website is available at <https://gitlab.igem.org/2022/sustech-shenzhen>.

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![About Image](https://static.igem.wiki/teams/4425/wiki/result.jpg)

## Results

## Plasmid Construction

Thanks to LINKS\_China 2021 team for providing us with part BBa\_K4011004(TnaA-FL-FMO) and part BBa\_K4011003(Fre-sttH).

![Blog One](https://static.igem.wiki/teams/4425/wiki/result1.png)
![Blog One](https://static.igem.wiki/teams/4425/wiki/result2.png)

We constructed plasmid pET28a-FMO-PtUGT to co-express PtUGT and FMO to convert indole or 6-Br-indole to indican or 6-Br-indican respectively. We also construct plasmid pET28a-BGL and pET28a-FMO to individually express BGL and FMO.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result3.png)
![Blog One](https://static.igem.wiki/teams/4425/wiki/result4.png)
![Blog One](https://static.igem.wiki/teams/4425/wiki/result5.png)

We transform part TnaA-FL-FMO and Fre-sttH into DH5α cell without endogenous TnaA gene. After culture and induced expression of these strains, we use the SDS-PAGE to separate and show the expression of TnaA-FL-FMO and Fre-sttH. We found that we successfully express our fused proteins and these two proteins show high water solubility.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result6.png)

##### FIGURE 1

 SDS-PAGE of TnaA-FL-FMO and Fre-sttH. WC stand for whole cell sample and S stand for supernatant sample after cell lysis.

We firstly constructed three plasmids pTrpR, pPOS and pNEG. pTrpR is induced to express muted trp repressors. Active trp repressors with its ligand can induce pPOS to express Taq polymerase and inactive trp repressors or trp repressor without its ligand can induce pNEG to express Taq polymerase.

For GFP test of CPR cycle, we constructed pNEG-sfGFP and pPOS-sfGFP which replace Taq polymerase gene with sfGFP to characterize the expression of Taq polymerase.

For the mock selection of CPR cycle, we inserted a DNA sequence of 200bp into wild type trp repressor to construct INAC-pTrpR (Figure 18). We mix plasmid pTrpR with wild type trp repressor and INAC-pTrpR with a mass ratio of 1:1 and transform the mixture into pNEG and pPOS competent cells. Induced cells were resuspend by CPR mix and emulsion droplets were constructed by mixing CPR mix and oil mix and vortex for 4 min.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result7.png)

##### FIGURE 2

 Scheme of compartmentalized partnered Replication (CPR) positive and negative circuits.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result8.png)

##### FIGURE 3

 Scheme of INAC-pTrpR construction

## Fermentation and dyeing

We then culture strain that has been induced to express Fre-sttH by NPB solution and 2.5 mM trp and 300 mM NaBr as substrate. During fermentation of 24 h, we use LC-MS to obtained data of concentration of trp and 6-Br-trp by every 6 h. Strain that has been induced to express TnaA-FL-FMO also be cultured by NPB solution and 1 mM trp or 6-Br-trp as substrate. After fermentation for 24 h, we get obvious blue and purple dye in our culture tubes . Besides, we attempt to produce tyrian purple by TnaA-FL-FMO using the culture after fermentation of trp and NaBr with Fre-sttH and the result has no difference from fermentation using standard 1 mM 6-Br-trp.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result9.png)

##### FIGURE 4

 Concentration-time graph of trp and 6-Br-trp in culture of 2.5mM trp and 300mM NaBr with Fre-sttH.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result10.png)

##### FIGURE 5

 Tyrian purple (left) and indigo (right) produced by 6-Br-trp and trp with TnaA-FL-FMO.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result11.png)

##### FIGURE 6

 Tyrian purple produced by using culture of fermentation with Fre-SttH and trp.

For purified indigo and tyrian purple, we dissolve the indigo from the cell pellet by DMSO and filter with a 0.2 μm strainer. By diluting DMSO with water, the pigment is precipitated from the solution to obtain a relatively pure indigo solid precipitation. We also use the tyrian purple we produce in cloth dying.

Dye concentration is calculated by a OD610/620 standard curve measured by 2021 iGEM team LINKS\_China. We get a ~60% yield for indigo and ~20% yield for tyrian purple.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result12.png)

##### FIGURE 7

 DMSO solution of indigo (top) and tyrian purple (bottom)

![Blog One](https://static.igem.wiki/teams/4425/wiki/result13.png)

##### FIGURE 8

 Relatively pure tyrian (left) and indigo (right) solid precipitation by diluting DMSO with water

![Blog One](https://static.igem.wiki/teams/4425/wiki/result14.png)

##### FIGURE 9

 Frabic dyed with tyrian purple

## Test of CPR

The expression of Taq polymerase cannot be monitored easily, we apply GFP assay to use GFP production to analyzing the gene circuits function. We also construct pNEG-sfGFP and pPOS-sfGFP which replace Taq polymerase gene with sfGFP to characterize the expression of Taq polymerase. We transform pTrpR which express wild type trp repressor into competent cells that have been transformed with plasmid pNEG-sfGFP or pPOS-sfGFP. Then we culture and induce with different concentration of trp and 1mM IPTG to induce expression of sfGFP. We expect that as trp concentration increases, the expression of sfGFP (RPU/OD600) in pNEG-sfGFP gradually decreases, and the expression of sfGFP (RPU/OD600) in pPOS-sfGFP gradually increases.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result15.png)

##### FIGURE 10

GFP production (RPU/OD600) -trp concentration graph by GFP test for wild type trp repressor and pNEG (due to time and pandemic, we only completed the experiment of pNEG).

In addition to GFP test, we also perform a mock selection to confirm the designed gene circuit function can actually select positive clone. At first, we mix culture with strains that has been induced to express sfGFP and oil mix to check the quality of emulsion droplet. We measure the diameter of droplet with a phase contrast microscopy. We found that the diameter of emulsion droplets are range about 2-10 μm and less than 20% emulsion droplets contain a GFP-expression cell. Thus, we have checked any emulsion droplet can only contain a single cell.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result16.png)

##### FIGURE 11

Phase contrast microscopy image of emulsion droplet of 50× diluted scale bar, 10 μm (left) of emusion droplet merge with GFP expression of 1× diluted emulsion. scar bar, 10 μm.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result17.png)

##### FIGURE 12

From left to right: cell culture emulsified with emulsion mix; Cell emulsion after ePCR circle; Emulsion after perform centrifuge; Phase-separate solution after breaking emulsion.

We insert a random sequence into wild type trp repressor to make it inactive to bind to trp, and we get plasmid INAC-pTrpR. We mix plasmid pTrpR with wild type trp repressor and INAC-pTrpR with a mass ratio of 1:1 and transform the mixture into pNEG and pPOS competent cells. A mock CPR seletion is next carried out. Induced strains is emulsified with emulsion mix and replicate positive clone by ePCR. The emulsion remains in a phase-independent state during ePCR. After breaking the emulsion by add ddH2O and chloroform, we apply a recovery PCR and perform a agarose electrophoresis to monitor the result the mock selection. PNEG give an obvious amplification of inactive trp repressor and pPOS give an obvious amplification of active trp repressor.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result18.png)

##### FIGURE 13

Result of agarose electrophoresis between trpR: INAC-trpR = 1:1 and trpR and inactive trpR after mock selection with pNEG (due to time and pandemic, we only completed the experiment of pNEG)

## Directed evolution of TrpR

Our libraries are designed in which residues (I57, V58, R54, L60, G85, T81, I82) that might contact trp at its 6C positions of the indole ring due to their proximity. Considering several position overlapped with those involved in the formation of the HTH motif, we initially choose I57, V58 and T81 as our mutant site for directed evolution. We perform site-directed saturation mutation on these three residues by completely randomized through re-synthesis of the gene with NNS codons (N = A, T, G, C; S = G, C). The library (32768 variants) are transformed into competent cells that have been transformed with pNEG or pPOS beforehand and carry out several circle of CPR to select positive clone we want. Due to the restriction of time, we haven’t perform a selective circle in our project, we plan to simulate this process using mathematical modeling.

![Blog One](https://static.igem.wiki/teams/4425/wiki/result19.png)

##### FIGURE 14

3D view of trpR binding to DNA and the trp binding site

#### Results

* [Plasmid Construction](#Plasmid Construction)
* [Fermentation and dyeing](#Fermentation and dyeing)
* [Test of CPR](#Test of CPR)
* [Directed evolution of TrpR](#Directed evolution of TrpR)

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© 2022 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
The repository used to create this website is available at <https://gitlab.igem.org/2022/sustech-shenzhen>.