iGEM 2022 — Team worldshaper-njbiox

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# Our Notebook

## March & April

###### Team Registration

###### Professional theory training online

## May

### Professional theory training: instrument design, media management, molecular biology

### Team leader selection

### Topic brainstorming

### Background survey

### Initiating fund raising

## June

* ##### Team division
* ##### Dry team

  #### Logo/ team jersey design

  #### Initiating fund raising

  #### self-media account operating (ins, youtube, Wechat ,bilibili)

  #### Developing online platform
* ##### Wet team

  #### Lab preparation

  #### Background description researching

  #### Expert interview

  #### Learning web page design

Since July, members finish the preparation work and devote their time into formal iGEM activities

## July

* ##### Wet team

  #### Starting lab

  #### Writing lab daily notes

  #### Modifying partnership line

  #### Handing out questionnaires

  #### Semi-structured interviews with professional researchers
* ##### Dry team

  #### Finishing interview outline

  #### Performing publicity propaganda

  #### making promotion video

  #### Starting to construct wiki framework

## August

* ##### Wet team

  #### lab experiments

  #### Writing lab design, protocol, and contribution

  #### Revising descriptions
* ##### Dry team

  #### Publicity events

  #### Popular science activities on social media

  #### Writing implementation

  #### Building up wiki web page

## September

* ##### Wet team

  #### lab experiments and finish

  #### Wiki manuscript
* ##### Dry team

  #### Meetup in Nanjing

  #### Online meetup

  #### Collaboration and partnership

  #### Modeling

  #### Hardware design

  #### Self-media update

  #### Wiki manuscript

## October

### Wiki manuscript

### Prepare for presentation

### Wiki web page optimization

### Prepare for presentation

### Parts registry

### Judging form

##### 2022 Timeline

###### [March & April](#March)

###### [May](#May)

###### [June](#June)

###### [July](#July)

###### [August](#August)

###### [September](#September)

###### [October](#October)

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# Engineering Success

## Abstract

Our team, the Worldshaper-NJBIOX, aims to use kitchen waste, shrimp and crab shells, as raw material to yield a biofuel, butyl butyrate, through the efficient biological production
of engineered Clostridium tyrobutyricum (C. tyrobutyricum) and a catalyzed reaction by a lipase from engineered E. coli.

#### In this project, our team has successfully made 9 parts to engineer C. tyrobutyricum and E. coli.

* 1) For the engineered C. tyrobutyricum, native C. tyrobutyricum contains a synthesis pathway of butyrate. An intermediate butyryl-CoA in this pathway can be catalyzed into butanol
  by aldol dehydrogenase (Figure 1). So, we inserted adhE2 to express aldehyde dehydrogenase. In addition, we enhanced the synthesis pathways of butyrate and butanol, by
  overexpressing thl and hbd, which encode two main enzymes, thiolysis enzyme and β-hydroxybutyryl-coenzyme, in the pathways, and by conjugating thiolysis enzyme (thl) and
  β-hydroxybutyryl-coenzyme (hbd) through a SpyTag/SpyCatcher system.
* 2) For the engineered E. coli, CALB gene was inserted to express CALB lipase from Candida antarcticum via a pet25b-T7-pelB-CALB-ChBD plasmid. By adding an affinity tag ChBD and
  pelB leader to CALB, we enhanced the reaction rate of CALB catalyzed esterification.

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/1.png)

### Figure 1 Synthesis pathway of butyrate and butanol by in our engineered C. tyrobutyricum

## Table 1 Part list

| No. | Name | Type | Description | Length |
| --- | --- | --- | --- | --- |
| 1 | BBa\_K4408004 | Scar | 19aa CALB-ChBD linker | 57 bp |
| 2 | BBa\_K4408005 | Composite | thl modified by oligopeptide SpyCatcher | 1569 bp |
| 3 | BBa\_K4408006 | Composite | hbd modified by oligopeptide SpyTag | 921 bp |
| 4 | BBa\_K4408007 | Composite | Lipase CALB connected with chitin binding domain | 1440 bp |
| 5 | BBa\_K4408008 | Device | Pthl-adhE2, Expression of adhE2 with Pthl promoter | 3244 bp |
| 6 | BBa\_K4408009 | Device | Pthl-adhE2-thl-hbd, Expression of adhE2, thl and hbd with Pthl promoter | 5309 bp |
| 7 | BBa\_K4408010 | Device | Pthl-adhE2-Spythl-Spyhbd, Expression of adhE2, thl and hbd,  modified by SpyCatcher/SpyTag oligopeptides | 5788 bp |
| 8 | BBa\_K4408011 | Device | T7-CALB-ChBD, Expression of CALB connected with chitin binding domain | 1363 bp |

## 1. Plasmid construction

### 1.1. Plasmids for C. tyrobutyricum

#### 1.1.1 adhE2 (aldehyde dehydrogenase) expression: pMTL-Pthl-adhE2 recombinant plasmid

We took a recombinant plasmid pMTL-Bs2 constructed previously by our research group as template, and used PCR to obtain a linearized pMTL-Pthl vector. adhE2 gene fragment was
amplified from the genome of Clostridium acetobutylicum by PCR. DNA electrophoresis confirmed the lengths of the two PCR products (5461bp, and 2577bp). adhE2 gene fragment was
ligated with pMTL-Pthl linearized vector into a pMTL-Pthl-adhE2 recombinant plasmid by Gibson assembly method. pMTL-Pthl-adhE2 was transformed into E. coli JM109 strain. Colony PCR
and DNA electrophoresis (750 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to
be pMTL-Pthl-adhE2 by gene sequencing.

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/2.png)

### Figure 2 Genetic circuit of pMTL-Pthl-adhE2

#### 1.1.2 Expression of adhE2 (aldehyde dehydrogenase), thl (thiolase) and hbd (β-hydroxybutyryl-coenzyme) : pMTL-Pthl-adhE2-thl-hbd recombinant plasmid

The recombinant plasmid pMTL-Pthl-adhE2 is cleaved using XbaⅠ enzyme to obtain the linear vector. The thl fragment (1182bp) and hbd fragment (849bp) were amplified from the C.
tyrobutyricum genome by PCR. The thl fragment was ligated to the linear vector pMTL-Pthl-adhE2 by Gibson assembly method. Similar as for pMTL-Pthl-adhE2, through transformation,
colony PCR and gene sequencing, we obtained the pMTL-Pthl-adhE2-thl recombinant plasmid and confirmed it. pMTL-Pthl-adhE2-thl was linearized by reverse PCR. The hbd fragment and the
linear vector pMTL-Pthl-adhE2-thl were ligated by Gibson assembly method. Using the same method as in 1.1.1, we obtained the pMTL-Pthl-adhE2-thl recombinant plasmid and confirmed it.
The recombinant plasmid pMTL-Pthl-adhE2-thl-hbd was also verified by enzymatic cleavage using Hind III enzyme.

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/3.png)

### Figure 3 Genetic circuit of pMTL-Pthl-adhE2-thl-hbd

#### 1.1.3 Conjugation of thiolysis (thl) and β-hydroxybutyryl-coenzyme (hbd) by a SpyTag/SpyCatcher system: pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd recombinant plasmids

pMTL-Pthl-adhE2 recombinant plasmid was digested with XbaI enzyme to obtain the linear vector. Using C. tyrobutyricum genome as template, PCR amplification was performed by
introducing SpyCatcher and SpyTag sequences through primers, resulting in fragment Spythl (1182bp) (i.e., SpyCatcher-thl) and fragment Spyhbd (849bp) (i.e., SpyTag-hbd). The fragment
Spythl was ligated with the linear vector pMTL-Pthl-adhE2 by Gibson assembly method. The correct pMTL-Pthl-adhE2-Spythl recombinant plasmid was obtained and verified by the same
method as in 1.1.

The linear vector pMTL-Pthl-adhE2-Spythl was obtained by reverse PCR, and ligated with the fragment Spyhbd by Gibson assembly method. The correct
pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd recombinant plasmid was obtained and verified by the same method as in 1.1.1. The recombinant plasmid was also verified by enzymatic
cleavage using Hind III enzyme.

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/4.png)

### Figure 4 pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/4-1.png)

### Figure 5 Gel electrophoresis results of colony PCR verification of pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd plasmid (1062 bp)

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/4-2.png)

### Figure 6 Gel electrophoresis results of enzymatic cleavage verification of pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd plasmid (2512 bp and 8011 bp)

### 1.2. Plasmid for E. coli: pet25b-T7-pelB-CALB-ChBD plasmid for the expression of lipase CALB

The linear vector pet25b was obtained by double digestion of pet25b using NdeI and EcoRⅠ enzymes. CALB fragment (966 bp) was amplified from the Pseudomonas aeruginosa genome by PCR.
ChBD fragment (417 bp) was obtained from a synthetic gene by PCR. The two fragments were fused together by fusion PCR, and then ligated to the linearized vector pet25b by T4
ligation. The pet25b-T7-pelB-CALB-ChBD recombinant plasmid was obtained and verified by the same method as in 1.1.1.

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/5.png)

### Figure 7 Genetic circuit of pet25b-T7-pelB-CALB-ChBD

## 2. Construction of engineered bacteria

### 2.1. Engineering C. tyrobutyricum

The three constructed plasmids for C. tyrobutyricum were conjugatively transferred to C. tyrobutyricum using E. coli CA434 as the donor strain, respectively, and the corresponding
engineered strains were achieved as following.

#### 2.1.1 Expression of adhE2 to establish a butanol synthesis pathway

The plasmid pMTL-Pthl-adhE2 was transferred to C. tyrobutyricum. Protein gel electrophoresis showed that aldehyde dehydrogenase (94 kDa) encoded by adhE2 was expressed in the
engineered C. tyrobutyricum (Figure 8). Fermentation experiment showed that adhE2 overexpression did not affect the growth of the strain (Figure 7).

HPLC experiment showed that 3.0 g/L butanol and 1.0 g/L butyrate were obtained from this strain, thus realizing a de novo synthesis pathway of butanol while maintaining the native
synthesis pathway of butyrate (Figure 10).

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/6.png)

### Figure 8 Protein gel electrophoresis of adhE2 expression in C. tyrobutyricum transformed with pMTL-Pthl-adhE2

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/7.png)

### Figure 9 Growth performance of recombinant plasmid pMTL-Pthl-adhE2 overexpression in C. tyrobutyricum

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/8.png)

### Figure 10 Butyrate and butanol yields of C. tyrobutyricum transformed with pMTL-Pthl-adhE2

#### 2.1.2 Overexpression of thl and hbd to optimize the butanol and butyrate synthesis pathways

The plasmid pMTL-Pthl-adhE2-thl-hbd was transformed into C. tyrobutyricum. Protein gel electrophoresis showed that thiolysis (thl) and β-hydroxybutyryl-coenzyme (hbd) were expressed
in the transformed strain (Figure 11). Fermentation experiment showed that the overexpression of these key enzymes effectively improved the growth performance of the strain, the
maximum OD600 increased by 21% (Figure 12).

HPLC experiment showed that the yields of butyrate and butanol were increased by 30% and 33%, respectively, in this strain (Figure 11). Since they are the precursors for butyl
butyrate synthesis catalyzed by lipase, the final butyl butyrate production was up to 590 mg/L in this strain, an increase by 1.1 fold compared to the strain without thl and hbd
overexpression (Figure 13).

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/9.png)

### Figure 11 Enhanced expression of thl and hbd in C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/10.png)

### Figure 12 Growth performance of C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/11.png)

### Figure 13 Yields of butyrate and butanol of C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/12.png)

### Figure 14 Butyl butyrate yield from C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd vs. that from C. tyrobutyricum transformed with pMTL-Pthl-adhE2

#### 2.1.3 Conjugation of thl and hbd via a SpyCatcher-SpyTag system to optimize the butanol and butyrate synthesis pathways

The plasmid pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd was transformed into C. tyrobutyricum. And the experiments are still in process.

### 2.2. Engineering E.coli: Expression of CALB lipase

The plasmid pet25b-T7-pelB-CALB-ChBD was transformed into E. coli Rosetta(DE3). Protein gel electrophoresis showed that E. coli was successfully constructed to produce Candida
antarctica Lipase B (CALB) (Figure 14).

CALB expressed by the plasmid has an affinity tag ChBD fused to it. ChBD-fusion CALB can facilitate the purification of CALB by chitin pellets, thus enhancing the reaction rate of
CALB catalyzed esterification.

In the enzyme activity assay, the exogenously expressed lipase was found to have good activity, showing the highest enzyme activity of 110 U/mL at 120 min (Figure 13). 220 mg/L butyl
butyrate was obtained by catalyzing C. tyrobutyricum transformed with pMTL-Pthl-adhE2. After immobilization by chitin pellets, the enzymatic activity of CALB increased by 24% to 136
U/mL, and the final catalytic butyl butyrate production was increased by 28% to 280 mg/L.

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/13.png)

### Figure 15 Lipase expression in E. coli

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/14.png)

### Figure 16 Enzyme activity assay of exogenously expressed lipase CALB

![blog](https://static.igem.wiki/teams/4408/wiki/engineering/15.png)

### Figure 17 Catalytic process of butyl butyrate synthesis

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