iGEM 2018 — Team SDU-CHINA

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:SDU-CHINA/Notebook

[![LOGO](https://static.igem.org/mediawiki/2018/7/72/T--SDU-China--duihui.jpg)](https://2018.igem.org/Team%3ASDU-CHINA)

* PROJECT ▾
  + [Description](https://2018.igem.org/Team%3ASDU-CHINA/Description)
  + [Design](https://2018.igem.org/Team%3ASDU-CHINA/Design)
  + [Results](https://2018.igem.org/Team%3ASDU-CHINA/Results)
  + [Demonstrate](https://2018.igem.org/Team%3ASDU-CHINA/Demonstrate)
* WETLAB ▾
  + [Protocols](https://2018.igem.org/Team%3ASDU-CHINA/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ASDU-CHINA/Notebook)
  + [Safety](https://2018.igem.org/Team%3ASDU-CHINA/Safety)
* DRYLAB ▾
  + [Model](https://2018.igem.org/Team%3ASDU-CHINA/Model)
  + [Hardware](https://2018.igem.org/Team%3ASDU-CHINA/Hardware)
* TEAM ▾
  + [Members](https://2018.igem.org/Team%3ASDU-CHINA/Team)
  + [Attributoins](https://2018.igem.org/Team%3ASDU-CHINA/Attributions)
  + [Collaborations](https://2018.igem.org/Team%3ASDU-CHINA/Collaborations)
* [INTERLAB](https://2018.igem.org/Team%3ASDU-CHINA/InterLab)
* PARTS ▾
  + [Parts](https://2018.igem.org/Team%3ASDU-CHINA/Parts)
  + [Improve](https://2018.igem.org/Team%3ASDU-CHINA/Improve)
* [HUMAN PRACTICES ▾](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices)
  + [Silver](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices#Silver)
  + [Gold & Integrated](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices#integrated)
  + [Engagement](https://2018.igem.org/Team%3ASDU-CHINA/Public_Engagement)

# Notebook

[week1](#week1)

[week2](#week2)

[week3](#week3)

[week4](#week4)

[week5](#week5)

[week6](#week6)

[week7](#week7)

[week8](#week8)

[week9](#week9)

[week10](#week9)

[week11](#week9)

[week12](#week12)

[week13](#week13)

[week14](#week14)

[week15](#week15)

[week16](#week16)

[week17](#week17)

[week18](#week18)

[week19](#week19)

[week20](#week20)

[week21](#week21)

[week22](#week22)

[week23](#week23)

#### Week 1 (April 25th to April 29th) [▲ back](#rili)

Plasmid backbones pSR43.6 and pSR58.6 were assembled by PCR and Gibson Assembly and the DNA fragments cloned from original plasmid pJ101700 and paracr15A was examined by electrophoresis. To test whether the backbones were assembled correctly, colony PCR and electrophoresis were required and then the backbones were send to sequencing.

![](https://static.igem.org/mediawiki/2018/0/0e/T--SDU-China--week11.png)

During electrophoresis of the PCR product, we discovered that the maker was not seen clearly.
We made Gibson Assemble: [Protocol 3](2018.igem.org/wiki/images/4/4c/T--SDU-China--Gibson_assembly_protocol.pdf)

|  |  |
| --- | --- |
| Assembly master mixture | 1.25 ml |
| 5×isothermal reaction buffer | 500μl |
| T5 exonuclease (10U/μl) | 1μl |
| DNA polymerase (2U/μl) | 31.25μl |
| Taq DNA ligase (40U/μl) | ddH2O |
| DNA fragment | Equal volume |

![](https://static.igem.org/mediawiki/2018/f/f3/T--SDU-China--week12.jpg)
![](https://static.igem.org/mediawiki/2018/0/06/T--SDU-China--week13.jpg)

![](https://static.igem.org/mediawiki/2018/6/67/T--SDU-China--week14.jpg)

All plasmid backbones of pSR58.6 showed in correct length except pSR58.6 NO.10.

#### Week 2 (May 2nd to May 8th) [▲ back](#rili)

We derived our plasmid backbones pSR43.6 and pSR58.6 and then sent them for synthesising the final plasmid.
To test the influence of blocking gltA to cell growth, we decided to construct a new plasmid p58.6gRNA containing Para and gRNA for type I-e CRISPRi and gltA.
DNA fragments were copied from pSR43.6 and pcrRNA-bssl-7(provided by our lab), the PCR products are shown below.

![](https://static.igem.org/mediawiki/2018/9/9d/T--SDU-China--week21.jpg)

New plasmid was assembled by Gibson Assembly, and transformed into *E. coli* DH5α, then colony PCR and electrophoresis were applied and we sent the prodcuts to sequencing.

![](https://static.igem.org/mediawiki/2018/c/c3/T--SDU-China--week22.jpg)

The result of electrophoresis showed only pSR58.6gRNA-8 was correct.
To assemble pcrRNA.bbsI-7-phac, we cloned DNA fragments from pcrRNA.bbsI-7 and pBHR68 by PCR, and the two fragments were assembled by Gibson assembly. But we failed in PCR, and it took us some time to repeat PCR.

![](https://static.igem.org/mediawiki/2018/9/90/T--SDU-China--week23.jpg)

Considering the failures in assembling pPT7-sfGFP, we cloned four DNA fragments (sfGFP、CmR、Ori、araR) by PCR and tried to assemble them by Gibson assembly at a time. Then this new plasmid was transformed into *E. coli* DH5α.

![](https://static.igem.org/mediawiki/2018/b/b8/T--SDU-China--week24.jpg)

#### Week 3 (May 9th to May 15th) [▲ back](#rili)

pSR58.6gRNA was transformed into *E. coli* EE-E15, in which Cas3 has been knocked out, provided by our lab.

![EEgRNA-5 was not correct](https://static.igem.org/mediawiki/2018/2/2d/T--SDU-China--week31.jpg)
(EEgRNA-5 was not correct)

After testing the transformation, growth curve was made to figure out the impact of crRNA along with CRISPRi.

![](https://static.igem.org/mediawiki/2018/b/b3/T--SDU-China--week32.jpg)
![](https://static.igem.org/mediawiki/2018/e/ea/T--SDU-China--week33.jpg)
(BC: blank control; c: arabinose without pSR58.6gRNA; a: arabinose and pSR58.6gRNA; concentration of arabinose: 0.2g/L; 0.4g/L; 0.6g/L)
![](https://static.igem.org/mediawiki/2018/4/4e/T--SDU-China--week34.jpg)
![](https://static.igem.org/mediawiki/2018/b/bf/T--SDU-China--week35.jpg)
(BC: blank control; c: arabinose without pSR58.6gRNA; a: arabinose and pSR58.6gRNA; concentration of arabinose: 0.02g/L; 0.2g/L; 2g/L)

As for pcrRNA.bbsI-7-phac, the PCR products were still incorrect tested by electrophoresis. So we tried several different PCR systems and redesigned primers.

pPT7-sfGFP was not correct by Gibson, we tried restriction sites and restriction enzymes instead, the new plasmid was transformed into *E. coli* DH5α and tested by colony PCR and sequencing.

#### Week 4 (May 16th to May 23rd) [▲ back](#rili)

We cultured bacteria transformed with pSR58.6gRNA in M9 (commonly used for industrial fermentation), and repeated measuring growth curve following the same protocol as week 3.

We discovered the impact of type I-e CRISPRi to bacteria growth is relatively weak.
pcrRNA.bbsI-7-phac was assembled by both Gibson assembly and restriction enzymes, and it was successfully assembled by Gibson assembly according to sequencing results.

![](https://static.igem.org/mediawiki/2018/3/39/T--SDU-China--week41.jpg)

#### Week 5 (May 24th to May 31st) [▲ back](#rili)

PT7 was inserted into pcrRNA.bbsI-7-phac through PCR and Gibson assembly, but it was failed. So we tried to assemble the plasmid by restriction enzymes.

![](https://static.igem.org/mediawiki/2018/9/98/T--SDU-China--week51.jpg)

#### Week 6 (June 4th to June 10th) [▲ back](#rili)

We were appreciated to receive pSR43.6r and pHZ from HZAU-China2017 and they were transformed into *E. coli* DH5α. pSR43.6r was derived and the ccaS component was improved by deleting about eight hundred base pairs according to paper [1]. But we were stopped by PCR.
pHZphbc, which contains phbCAB gene cluster, was assembled by PCR and Gibson assembly.

![](https://static.igem.org/mediawiki/2018/3/3b/T--SDU-China--week61.jpg)

phbCAB gene cluster was inserted into pcrRNA.bbsI-7 by both restriction enzymes and Gibson assembly. The sequencing result showed that this new plasmid was assembled successfully by Gibson.

![](https://static.igem.org/mediawiki/2018/a/ae/T--SDU-China--week62.jpg)

[1]. Nakajima, M., et al., Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal. Scientific Reports, 2016. 6(1).

#### Week 7 (June 11th to June 17th) [▲ back](#rili)

pHZphbc was failed in assebling, so we repeated cloning and redesigned the plasmid at the same time. Tested by colony PCR and sequencing, pSR43.6-ccaS#10 was successfully assembled. To insert crRNA into pSR43.6r, we needed to construct an intermediate plasmid called pHZZJ.

pPT7-crRNA was assembled successfully by restriction enzymes after editing PT7 for a long time.

#### Week 8 (June 18th to June 24th) [▲ back](#rili)

pHZphbc and pHZZJ were assembled successfully. To construct pSR43.6-ccaS#crRNA, DNA fragments were copied from pSR43.6-ccaS#10 and pHZZJ and then they were assembled through Gibson assembly.

pcrRNA.bbsI-7-opto-phbc was transformed into *E. coli* DE3, we wanted to test the production of PHB by Nile Red. We did this experiment applying [Protocol 15](https://static.igem.org/mediawiki/2018/d/d3/T--SDU-China--Nile_Red.pdf). Here is the result.

![](https://static.igem.org/mediawiki/2018/4/45/T--SDU-China--week81.png)

pPT7-sfGFP was assembled successfully by restriction enzymes.

#### Week 9-11 (June 25th to July 14th)[▲ back](#rili)

It was time for final examination!

#### Week 12 (July 15th to July 22th)[▲ back](#rili)

We were blocked by assembling pSR43.6-ccaS#10-crRNA, we tried both Gibson assembly and restriction enzyme, and we redesigned the plasmid and primers for many times. We had to figure out what was going on.
At the same time, pHZccdA which contains antitoxic protein ccdA but without promoter was assembled by PCR and Gibson assembly.

#### Week 13 (July 23rd to July 30th)[▲ back](#rili)

pSR43.6-ccaS#10-crRNA was still under assembling, we suspected that the leakage of crRNA might prevent bacteria from growing, so the Gibson product was transformed into EE-E15, in which Cas3 protein has been knocked out. EE-E15 grew at last but the result of colony PCR was incorrect.

Since the result of Nile Red wasn’t desirable, we tried to change the terminator of phbCAB gene cluster through restriction enzymes, but we failed.

#### Week 14 (July 31st to August 5th)[▲ back](#rili)

Promoters with different strength (J23101, J23105, J23108, J23113 and J23114) were inserted into pHZccdA through Gibson assembly. But only J23108 and J23113 were inserted successfully.
We tested the production of PHB with Nile Red again, according to [Protocol 15](https://static.igem.org/mediawiki/2018/d/d3/T--SDU-China--Nile_Red.pdf)
Result:

![](https://static.igem.org/mediawiki/2018/4/4e/T--SDU-China--week141.png)

#### Week 15 (August 6th to August 12th)[▲ back](#rili)

While assembling pHZJ23ccdA series, we characterized the improved ccaS-ccaR two-component system by measuring GFP fluorescence according to [Protocol 11](https://static.igem.org/mediawiki/2018/3/35/T--SDU-China--Characterization_of_CcaS-CcaR_system.pdf)
Result:

![](https://static.igem.org/mediawiki/2018/3/30/T--SDU-China--week151.png)

ccdB and degradation tag was inserted into pcrRNA.bbsI-7 after promoter T7 by Gibson assembly and restriction enzymes.

#### Week 16 (August 13th to August 20th)[▲ back](#rili)

We found that there were some problems in pSR43.6-ccaS#10 by characterizing pSR43,6r and pHZ, so we redesigned this plasmid as well as other pSR43.6-ccaS# series (#3,#4), we assembled pSR43.6-ccaS# series by PCR and Gibson assembly. pSR43.6-ccaS# series were assembled successfully.
Result for characterization:

![](https://static.igem.org/mediawiki/2018/c/c9/T--SDU-China--week161.png)

The new plasmid pcrRNA.bbsI-7-ccdb was assembled and tested by colony PCR and sequencing.

The split T7 RNA polymerase was characterized under blue light according to [Protocol 13](https://static.igem.org/mediawiki/2018/b/bb/T--SDU-China--Characterization_of_split_RNAP.pdf)

The result showed that the split T7 RNA polymerase did not work, we held a troubleshooting for that with our advisors and instructors, wanting to figure out the problems and solutions.

#### Week 17 (August 21st to August 28th)[▲ back](#rili)

After finishing assembling pSR43.6-ccaS# series, they were transformed into *E. coli* TOP10 along with pHZ, and the ccaS-ccaR two-component system was characterized again according to [Protocol 11](https://static.igem.org/mediawiki/2018/3/35/T--SDU-China--Characterization_of_CcaS-CcaR_system.pdf).

Finally pSR43.6-ccaS#-crRNA series were born through Gibson assembly.

we wondered if there was something wrong with PT7 and split RNA polymerase, so pET28a(DE3) was applied to test whether pT7 could work properly, and we did SDS-PAGE to detect the expression of proteins. But pT7 worked properly.

![](https://static.igem.org/mediawiki/2018/4/45/T--SDU-China--week171.png)

#### Week 18 (August 29th to September 7th)[▲ back](#rili)

pHZJ23ccdAB series were assembled by PCR and Gibson assembly.
pSR43.6-ccaS#-crRNA and pHZphbc were characterized by measuring the production of PHB according to [Protocol 14](https://static.igem.org/mediawiki/2018/8/83/T--SDU-China--Measurement_of_PHB_production-GC.pdf).

Blue light components were inserted into a high-copy plasmid backbone with high GFP expression, and this system was tested again under blue light according to [Protocol 13](https://static.igem.org/mediawiki/2018/b/bb/T--SDU-China--Characterization_of_split_RNAP.pdf).

We couldn’t figure out the problem of this system and we decided to stop experiment on blue light component due to the limitation of time.

#### Week 19 (September 10th to September 16th)[▲ back](#rili)

To make phbCAB gene cluster express constitutively, phbCAB gene cluster was inserted into pHZ3.1 through Gibson Assembly.
Ccas-ccaR system was characterized by measuring fluorescence, and production of PHB was measured according to [Protocol 13](https://static.igem.org/mediawiki/2018/b/bb/T--SDU-China--Characterization_of_split_RNAP.pdf).

![](https://static.igem.org/mediawiki/2018/9/94/T--SDU-China--week191.png)
![](https://static.igem.org/mediawiki/2018/6/68/T--SDU-China--week192.png)

Inhibition of bacteria growth by type I-e CRISPRi was tested by measuring cell density, but the result was not good enough.

#### Week 20 (September 17th to September 23rd)[▲ back](#rili)

We found two mutations in phbCAB gene cluster by sequencing, and we wondered it might be the cause of the low production of PHB. At the same time, plasmids that would be used as control were under assembling.

#### Week 21 (September 24th to October 1st)[▲ back](#rili)

We were busy with standardization and part registry, at the same time, bacteria strain responsible for production of PHB induced by green light was under constructing. We were still struggling testing the effect caused by crRNA to bacteria growth. Characterization of Ccas-CcaR system was done in 24-well plate. At the end of this week, part BBa\_K2627000 is successfully assembled and finished sequencing.

![](https://static.igem.org/mediawiki/2018/3/36/T--SDU-China--week211.png)

#### Week 22 (October 2nd to October 7th)[▲ back](#rili)

We were happy with the National Day Holiday, but we still need to continue our experiment or we might leave the project unfinished. Bacteria strains which can synthesize PHB induced by light were selected through Nile Red according to https://static.igem.org/mediawiki/2018/d/d3/T--SDU-China--Nile\_Red.pdf. Characterization of Ccas-CcaR system was done in 24-well plate, and characterization of crRNA was going not very well. But the good news was that at the end of this vocation, the rest of the parts we were willing to send were assembled and sequenced.

![](https://static.igem.org/mediawiki/2018/3/35/T--SDU-China--week221.png)
![](https://static.igem.org/mediawiki/2018/c/c3/T--SDU-China--week222.png)

#### Week 23 (October 8th to October15th)[▲ back](#rili)

Finally, we finished constructing the bacteria strain that could produce PHB under green light, and the new strain was tested by measuring the relative weight of PHB through Gas Chromatography and HPLC following the [protoco](https://static.igem.org/mediawiki/2018/8/83/T--SDU-China--Measurement_of_PHB_production-GC.pdf)l. Since the effect of type I-E CRISPRi was not ideal when induced by light, so we chose to test it with arabinose as its inducer.

![SPONSORS](https://static.igem.org/mediawiki/2018/a/ab/T--SDU-CHINA--Sponsors0.svg)

mailto:sdubioigem@163.com

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[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:SDU-CHINA/Experiments

[![LOGO](https://static.igem.org/mediawiki/2018/7/72/T--SDU-China--duihui.jpg)](https://2018.igem.org/Team%3ASDU-CHINA)

* PROJECT ▾
  + [Description](https://2018.igem.org/Team%3ASDU-CHINA/Description)
  + [Design](https://2018.igem.org/Team%3ASDU-CHINA/Design)
  + [Results](https://2018.igem.org/Team%3ASDU-CHINA/Results)
  + [Demonstrate](https://2018.igem.org/Team%3ASDU-CHINA/Demonstrate)
* WETLAB ▾
  + Protocols
  + [Notebook](https://2018.igem.org/Team%3ASDU-CHINA/Notebook)
  + [Safety](https://2018.igem.org/Team%3ASDU-CHINA/Safety)
* DRYLAB ▾
  + [Model](https://2018.igem.org/Team%3ASDU-CHINA/Model)
  + [Hardware](https://2018.igem.org/Team%3ASDU-CHINA/Hardware)
* TEAM ▾
  + [Members](https://2018.igem.org/Team%3ASDU-CHINA/Team)
  + [Attributoins](https://2018.igem.org/Team%3ASDU-CHINA/Attributions)
  + [Collaborations](https://2018.igem.org/Team%3ASDU-CHINA/Collaborations)
* [INTERLAB](https://2018.igem.org/Team%3ASDU-CHINA/InterLab)
* PARTS ▾
  + [Parts](https://2018.igem.org/Team%3ASDU-CHINA/Parts)
  + [Improve](https://2018.igem.org/Team%3ASDU-CHINA/Improve)
* [HUMAN PRACTICES ▾](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices)
  + [Silver](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices#Silver)
  + [Gold & Integrated](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices#integrated)
  + [Engagement](https://2018.igem.org/Team%3ASDU-CHINA/Public_Engagement)

# Protocols

#### [**Transformation**](#collapseOne)

***E. coli* transformation protocol**
Please click on link to view the [protocol for *E. coli* transformation](https://static.igem.org/mediawiki/2018/a/a4/T--SDU-China--E._coli_transformation_protocol.pdf).

#### [**Gel extraction**](#collapseTwo)

**Gel extraction protocol**
Please click on link to view the [protocol for gel extraction protocol](2018.igem.org/wiki/images/6/6a/T--SDU-China--gel_extraction_protocol.pdf).

#### [**Gibson assembly**](#collapseThree)

**Gibson assembly protocol**
Please click on link to view the [protocol for gibson assembly](2018.igem.org/wiki/images/4/4c/T--SDU-China--Gibson_assembly_protocol.pdf).

#### [**Gel electrophoresis**](#collapseFour)

**Agarose gel electrophoresis protocol**
Please click on link to view the [protocol for gel electrophoresis](https://static.igem.org/mediawiki/2018/9/92/T--SDU-China--agarose_gel_electrophoresis_protocol.pdf.pdf).

#### [**PCR(Vazyme)**](#collapseFive)

**PCR protocol(Vazyme)**
Please click on link to view the [protocol for PCR(Vazyme)](https://static.igem.org/mediawiki/2018/0/0e/T--SDU-China--vazyme_PCR_protocol.pdf).

#### [**PCR(Trans)**](#collapseSix)

**PCR protocol(Trans)**
Please click on link to view the [protocol for PCR(Trans)](https://static.igem.org/mediawiki/2018/0/05/T--SDU-China--Trans_PCR_protocol.pdf).

#### [**ABM assembly**](#collapseSeven)

**ABM assembly protocol**
Please click on link to view the [protocol for ABM assembly](https://static.igem.org/mediawiki/2018/9/9a/T--SDU-China--abm_assembly.pdf).

#### [**Preparation of competent cells**](#collapseEight)

**Protocol of preparing competent cells**
Please click on link to view the [protocol for preparing competent cells](https://static.igem.org/mediawiki/2018/9/91/T--SDU-China--Preparation_of_competent_cells.pdf).

#### [**Purification of bacteria plasmids**](#collapseNine)

**Protocol of purifying bacteria plasmids**
Please click on link to view the [protocol for purifying bacteria plasmids](https://static.igem.org/mediawiki/2018/7/7f/T--SDU-China--Purification_of_bacteria_plasmids_protocol.pdf).

#### [**PCR extraction**](#collapseTen)

**PCR extraction protocol**
Please click on link to view the [protocol for PCR extraction](https://static.igem.org/mediawiki/2018/6/6a/T--SDU-China--PCR_extraction_protocol.pdf).

#### [**Characterization of CcaS-CcaR system**](#collapseEleven)

**Characterization of CcaS-CcaR system**
Please click on link to view the [protocol for charactrizing CcaS-CcaR system](2018.igem.org/wiki/images/d/d4/T--SDU-China--characterization_of_CcaS-CcaR_system.pdf).

#### [**Characterization of crRNA**](#collapseTwelve)

**Characterization of crRNA**
Please click on link to view the  [charactrizing crRNA](https://static.igem.org/mediawiki/2018/c/c9/T--SDU-China--Characterization_of_crRNA.pdf).

#### [**Characterization of split RNAP**](#collapseThirteen)

**Characterization of split RNAP**
Please click on link to view the [protocol for charactrizing split RNAP](https://static.igem.org/mediawiki/2018/b/bb/T--SDU-China--Characterization_of_split_RNAP.pdf).

#### [**Measurement of PHB production**](#collapseFourteen)

**Measurement of PHB production**
Please click on link to view the [protocol for measuring PHB production](https://static.igem.org/mediawiki/2018/8/83/T--SDU-China--Measurement_of_PHB_production-GC.pdf).

#### [**Nile red stain**](#collapseFifteen)

**Nile red stain protocol**
Please click on link to view the [protocol for Nile red stain](https://static.igem.org/mediawiki/2018/d/d3/T--SDU-China--Nile_Red.pdf).

![SPONSORS](https://static.igem.org/mediawiki/2018/a/ab/T--SDU-CHINA--Sponsors0.svg)

mailto:sdubioigem@163.com

[www.instagram.com/sdubiochina\_2018/](https://www.instagram.com/sdubiochina_2018/)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:SDU-CHINA/Results

[![LOGO](https://static.igem.org/mediawiki/2018/7/72/T--SDU-China--duihui.jpg)](https://2018.igem.org/Team%3ASDU-CHINA)

* PROJECT ▾
  + [Description](https://2018.igem.org/Team%3ASDU-CHINA/Description)
  + [Design](https://2018.igem.org/Team%3ASDU-CHINA/Design)
  + [Results](https://2018.igem.org/Team%3ASDU-CHINA/Results)
  + [Demonstrate](https://2018.igem.org/Team%3ASDU-CHINA/Demonstrate)
* WETLAB ▾
  + Protocols
  + [Notebook](https://2018.igem.org/Team%3ASDU-CHINA/Notebook)
  + [Safety](https://2018.igem.org/Team%3ASDU-CHINA/Safety)
* DRYLAB ▾
  + [Model](https://2018.igem.org/Team%3ASDU-CHINA/Model)
  + [Hardware](https://2018.igem.org/Team%3ASDU-CHINA/Hardware)
* TEAM ▾
  + [Members](https://2018.igem.org/Team%3ASDU-CHINA/Team)
  + [Attributoins](https://2018.igem.org/Team%3ASDU-CHINA/Attributions)
  + [Collaborations](https://2018.igem.org/Team%3ASDU-CHINA/Collaborations)
* [INTERLAB](https://2018.igem.org/Team%3ASDU-CHINA/InterLab)
* PARTS ▾
  + [Parts](https://2018.igem.org/Team%3ASDU-CHINA/Parts)
  + [Improve](https://2018.igem.org/Team%3ASDU-CHINA/Improve)
* [HUMAN PRACTICES ▾](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices)
  + [Silver](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices#Silver)
  + [Gold & Integrated](https://2018.igem.org/Team%3ASDU-CHINA/Human_Practices#integrated)
  + [Engagement](https://2018.igem.org/Team%3ASDU-CHINA/Public_Engagement)

# Results

#### Attempt to build the split RNAP

We initially chose a blue-light-controlled sensor as candidate. The sensor, opto T7 RNA polymerase is a pair of fusion proteins which initiates transcription of PT7 by dimerize when exposed to light 450 nm. nMag is fused with the CT (carbon terminal)[1] of T7RNAP and pMag is fused with NT (nitrogen terminal). Due to the toxicity of the part, the expression of both the two fusion proteins are induced by L-arabinose[2]. And we set a concentration gradient to explore the optimized condition.

#### 1. Characterization of sensor opto T7 RNAP

We inserted the sfGFP after the PT7 to characterize the behavior of the blue light sensor. The experimental result shows no significant induction compared with the dark control.

![Figure 1. Dynamic experiment of the expression level of Opto-T7RNAP. The sensor is exposed to blue light and dark respectively at 0h. Result showed no significant expression of sfGFP.](https://static.igem.org/mediawiki/2018/e/e3/T--SDU-CHINA--result2.png "Figure 2. Dynamic experiment of the expression level of Opto-T7RNAP. The sensor is exposed to blue light and dark respectively at 0h. Result showed no significant expression of sfGFP.")

Figure 1. Dynamic experiment of the expression level of Opto-T7RNAP. The sensor is exposed to blue light and dark respectively at 0h. Result showed no significant expression of sfGFP.

#### 2.Validation at protein level

To verify the expression of the sensors, whose MWs are 53.9kD and 78.5kD respectively. We tested the sample containing sensor induced by 0, 0.1%, 0.2%, 0.5% arabinose and empty vector. The SDS-PAGE result shows no significant existence of the sensors.

![Figure 2. Analysis sensor expression in total E. coli protein by SDS PAGE.](https://static.igem.org/mediawiki/2018/6/60/T--SDU-CHINA--result3.png "Figure 2. Analysis sensor expression in total E. coli protein by SDS PAGE.")

Figure 2. Analysis sensor expression in total E. coli protein by SDS PAGE.

#### Characterization of CcaS/CcaR system

The CcaS protein acts as a light sensing part in CcaS/CcaR two-component system (TCS). CcaS consists of an N-terminal transmembrane helix; a GAF domain, which serves as the sensor domain; a linker region (L1); two PAS domains; a second linker region (L2); and a C-terminal histidine kinase (HK) domain. We were inspired by Professor Koji’s work[3] to remove the L1 linker region and the two PAS domains, then fuse the GAF and HK domains with a truncated linker region.

![Figure 3. Domain organization of wild-type CcaS and miniaturized CcaSs.](https://static.igem.org/mediawiki/2018/d/d1/T--SDU-CHINA--result4.png "Figure 3. Domain organization of wild-type CcaS and miniaturized CcaSs.")

Figure 3. Domain organization of wild-type CcaS and miniaturized CcaSs.

Our advanced system was characterized over wild type in M9 medium containing 2% glucose but without yeast extract. The fluorescence intensity was acquired 9 hours after inoculated into 24-well plate with Abs600 about 0.1. Our culture was exposed to different light to characterize different transcription level. The basic OD and fluorescence were wiped off. (n=3)

![Figure 4. This figure shows the fluorescence of cells grown under green light (green bars), red light (red bars), and darkness (gray). Bars indicate relative fluorescence intensity to normalized fluorescence level of wild type. Data represent means ±SD from three repeat experiment (n=3).](https://static.igem.org/mediawiki/2018/e/e6/T--SDU-CHINA--result5.png "Figure 4. This figure shows the fluorescence of cells grown under green light (green bars), red light (red bars), and darkness (gray). Bars indicate relative fluorescence intensity to normalized fluorescence level of wild type. Data represent means ±SD from three repeat experiment (n=3).")

Figure 4. This figure shows the fluorescence of cells grown under green light (green bars), red light (red bars). Bars indicate relative fluorescence intensity to normalized fluorescence level of wild type. Data represent means ±SD from three repeat experiment (n=3).

The dynamic ranges of green over red light of different system are: 2.8(wild type), 2.7(#3), 4.9(#10). The #4 has the reverse function and the dynamic range is about 1.5.

#### The construction of the metabolic flux regulation platform.

We selected type I-E CRISPRi system as the cut-over dynamically switching the metabolic flux. Type I-E CRISPRi system can inhibit the aimed gene’s transcription through expressing crRNA combining endogenous Cas protein.

#### 1. Characterization of type I-E CRISPRi system

To remove the DNA degradation function and maintain the DNA binding function of the modified type I-E CRISPR system, a special *E.coli* TOP10 with Cas3 protein knocked out and promoter of Cascade substituted by promoter J23119 was constructed and named EE-E15. To verify the system in EE-E15, the GFP gene was chosen as a reporter to test the function of crRNA targeting different sites of GFP*[4]*. The results showed that there were wide rages of repression among different sites and the spacer posited on the promotor region presented high repression level (82%).

![Figure 5. The site of spacers target gfp.](https://static.igem.org/mediawiki/2018/2/20/T--SDU-CHINA--result6.png "Figure 5. The site of spacers target gfp.")

Figure 5. The site of spacers target gfp.

![Figure 6. Characterization of type I-E CRISPRi system targeting different sites of gfp.](https://static.igem.org/mediawiki/2018/3/3e/T--SDU-CHINA--result7.png "Figure 6. Characterization of type I-E CRISPRi system targeting different sites of gfp.")

Figure 6. Characterization of type I-E CRISPRi system targeting different sites of gfp.

TCA cycle is one of the most significant metabolic pathways for energy supplement. The cycle starts with the process that citrate synthase catalyzes acetyl-coA and oxaloacetate to form citric, which is the rate-limiting step in the TCA cycle and irreversible, thus the regulation of expression of *gltA* which codes the citrate synthase may have great effect in central metabolism, and through model we also found that control of *gltA* can regulate the metabolic flux. We got the target site of *gltA* gene from previous work and then constructed a medium-copy plasmid (Paracr15A) and a high-copy plasmid (Paracr58.6) containing crRNA targeting *gltA* to inhibit expression. With 0.2% L-arabinose induction, the samples showed an obvious repression on growth compared with those without L-arabinose, regardless of the copy number difference between Paracr15A and Paracr58.6(Figure 8). Among them, aracr58.6 grew slower, which may due to its high leakage with high-copy plasmids. It indicated that this Type-I-E CRISPRi system can affect cell growth through producing crRNA targeting *gltA*.

![Figure 7. The posited site of crRNA targeting gltA to inhibit it’s transcription.](https://static.igem.org/mediawiki/2018/4/46/T--SDU-CHINA--result8.png "Figure 7. The posited site of crRNA targeting gltA to inhibit it’s transcription.")

Figure 7. The posited site of crRNA targeting *gltA* to inhibit it’s transcription.

![Figure 8. Regulation effect of crRNA induced by L-arabinose between medium-copy and high-copy plasmids. The strains were cultured in 24-well plate in 1 ml M9 medium containing 1% glycerol.](https://static.igem.org/mediawiki/2018/f/fe/T--SDU-CHINA--arabinosechanged.png "Figure 8. Regulation effect of crRNA induced by L-arabinose between medium-copy and high-copy plasmids. The strains were cultured in 24-well plate in 1 ml M9 medium containing 1% glycerol.")

Figure 8. Regulation effect of crRNA induced by L-arabinose between medium-copy and high-copy plasmids. The strains were cultured in 24-well plate in 1 ml M9 medium containing 1% glycerol.

#### 2. light-induced type I-E CRISPRi system

Further, we utilized CcaS/CcaR sensor with type I-E CRISPRi system to induction of crRNA targeting the site of *gltA* through switching light. To optimize this system, we chose different culture medium verifying the function of light-induced crRNA.

![Figure 9. Comparison of the regulation effects between different carbon sources. Strains were cultured in 1ml M9 medium containing 1% glucose in a) while cultured in M9 medium with 1% glycerol in b) in 24-well plates.](https://static.igem.org/mediawiki/2018/7/7d/T--SDU-CHINA--result10.png "Figure 9. Comparison of the regulation effects between different carbon sources. Strains were cultured in 1ml M9 medium containing 1% glucose in a) while cultured in M9 medium with 1% glycerol in b) in 24-well plates.")
![Figure 9. Comparison of the regulation effects between different carbon sources. Strains were cultured in 1ml M9 medium containing 1% glucose in a) while cultured in M9 medium with 1% glycerol in b) in 24-well plates.](https://static.igem.org/mediawiki/2018/d/da/T--SDU-CHINA--result11.png "Figure 9. Comparison of the regulation effects between different carbon sources. Strains were cultured in 1ml M9 medium containing 1% glucose in a) while cultured in M9 medium with 1% glycerol in b) in 24-well plates.")

Figure 9. Comparison of the regulation effects between different carbon sources. Strains were cultured in 1ml M9 medium containing 1% glucose in a) while cultured in M9 medium with 1% glycerol in b) in 24-well plates.

In the figures we can find that cell cultured with glycerol growth had a higher level of repression compared with glucose. It indicates that crRNA targeting *gltA* to inhibit TCA cycle from the first step showed a better effect when nutrition was not directly utilized. In addition, it illustrates that condition made sense for the function of our system. Meanwhile the phenomenon was analyzed through [flux balance analysis (FBA) mathematical model](https://2018.igem.org/Team%3ASDU-CHINA/Model). With the data we found there was a threshold when inhibiting *gltA*, exceeding which appears an obvious transform in metabolic flux. We analyzed that glycerol as carbon source can reduce this threshold showing great repression which was in accordance with the growth results described above. Then we chose M9 medium with 1% glycerol as the condition for the characterization of crRNA targeting *gltA*.

To optimize the expression of crRNAs in EE-E15, then we constructed a medium-copy plasmid (pSR43.6) and high-copy plasmid (phz) to express crRNA targeting endogenous *gltA*. The two strains showed significant variance in growth in figure 4, suggesting crRNA on high-copy plasmid showed stronger repression than on medium-copy one. We supposed that the inhibition function was related to the quantity of crRNA and light-sensing promotor PcpG2-172 works well in high-copy number, leading to this result. It indicated that copy-number for light-induced crRNA had a great impact on this photoactivated regulation system.

![Figure 10. Comparison of the regulatory effect between medium-copy and high-copy plasmids. #10 43.6 is a medium-copy plasmid (p15A ori) expressing crRNA while high-copy for #10 phz (CoIE1). The strains were cultured in 24-well plate in 1ml M9 medium containing 1% glycerol.](https://static.igem.org/mediawiki/2018/f/fe/T--SDU-CHINA--result12.png "Figure 10. Comparison of the regulatory effect between medium-copy and high-copy plasmids. #10 43.6 is a medium-copy plasmid (p15A ori) expressing crRNA while high-copy for #10 phz (CoIE1). The strains were cultured in 24-well plate in 1ml M9 medium containing 1% glycerol.")

Figure 10. Comparison of the regulatory effect between medium-copy and high-copy plasmids. #10 43.6 is a medium-copy plasmid (p15A ori) expressing crRNA while high-copy for #10 phz (CoIE1). The strains were cultured in 24-well plate in 1ml M9 medium containing 1% glycerol.

After the verification of the culture condition, we investigated the dynamic regulation effects of targeting *gltA* with light-controlled system through switching red light to green light at 2h, 5h and 9h. As shown in figure 5, all strains induced at different time grow better compared with illuminating at 0h. Bacteria growth was inhibited the most when being induced at 5h, showing an obvious palliation after sensing green light. The strains induced at the early-log phase (2h) grew slower than the control (upon red illumination), whereas it continuously grew and maintained a relatively high K value. It was supposed that cells growing at a moderate speed at early consumed less nutrition, thus drove the biomass constantly increase and had a higher level of K value. In accordance with the hypothesis, we concluded the negative control without crRNA targeting *gltA* which presented a lowest level of K value consumed too much nutrition so that it can’t satisfy the need of cell in late period of growth. In addition, inducing crRNA expression at 9h through green light, we found there were little growth variance between the strains green light induced at 9h and the control illuminated at red light continuously. It was supposed that at the stationary phase there was nearly no effect for inhibiting TCA cycle to repress the cell’s growth, which might due to its metabolic flow. The difference between the regulation effect of different induction time isn’t obvious through light switching, might due to light sensor CcaS/CcaR system which only have 4.8-fold difference between red/green light induction.

![Figure 11. Regulation effect of crRNA induced at different time by switching to green light. The strains were cultured in 50ml M9 medium containing 1% glycerol.](https://static.igem.org/mediawiki/2018/c/c4/T--SDU-CHINA--result13.png)

Figure 11. Regulation effect of crRNA induced at different time by switching to green light. The strains were cultured in 50ml M9 medium containing 1% glycerol.

We reconstructed EE-E15 driving its metabolic flux regulation at different time through switching red light to the green one. Then we made polyhydroxybutyrate(PHB) as an example to demonstrate our dynamically regulating metabolic flux platform.

#### PHB fermentation

#### 1. PHB synthesis with CcaS/CcaR system

Firstly, in order to verify that phbCAB cluster could be expressed induced by CcaS/CcaR system and synthesize Polyhydroxybutyrate (PHB) successfully, phbCAB cluster (without its original constitutive promoter) was cloned right behind the light sensing promoter, PcpcG2-172 (Figure 12).

![](https://static.igem.org/mediawiki/2018/a/a2/T--SDU-CHINA--result15.png "Figure 12. Gene circuit of PHB induced” alt=")

Figure 12. Gene circuit of "PHB induced”

Also strain “PHB induced Red/Green” was illuminated with red light and green light respectively throughout the PHB fermentation while light illuminating “PHB induced 5h” was switched from red light to green light at 5h (exponential phase) so that we can tell whether turning on PHB synthesis pathway at different periods could affect PHB content within the cells and cell growth, as well.

![](https://static.igem.org/mediawiki/2018/1/12/T--SDU-CHINA--result16.png)
![](https://static.igem.org/mediawiki/2018/4/4a/T--SDU-CHINA--result17.png)
![](https://static.igem.org/mediawiki/2018/c/cf/T--SDU-CHINA--result18.png)

Figure 13.Cell growth curve and PHB production among strain “PHB induced”. In groups "PHB induced 5h/Green", the expression of phbCAB is activated at 0h and 5h respectively. In contrast "PHB induced Red" are illuminated by red light throughout the fermentation to inhibit the crRNA expression. (a) cell growth curve during the PHB fermentation. (b) the ratio between PHB and cell drought weight, showing the average PHB content within each cell. (c) total amount of PHB produced during the fermentation.

According to the PHB fermentation results, CcaS/CcaR system expressed phbCAB cluster successfully (Figure 13.b c). What’s more, a large amount of PHB could be synthesized as PHB took up over 30% of cell drought at the end of fermentation in “PHB induced Green" (Figure 13.b). Furthermore, the amount of PHB inside the cells increased as the amount of time for phbCAB cluster expression increased, which was consistent with our expectation (Figure 13.b). However, to our surprise, it appeared that those groups containing higher PHB content tended to grow better than those with less PHB. After searching articles on the relationship between PHB synthesis and bacteria stress tolerance, we found out that the biosynthesized PHB stored inside the cell can serve as an intracellular energy and carbon storage system and thus, increases the cell survival rate when carbon source is limited.[5] This probably explains the reason why the “PHB induced” that synthesized more PHB tend to have higher cell density for the duration of fermentation.

#### 2. Effect of metabolic flux redirection on PHB production Since we have demonstrated that a great deal of PHB can be synthesized under the control of CcaS/CcaA system, we then hope to verify that the inhibition of *gltA* gene by crRNA as well as type I-E CRISPRi system can improve PHB production through metabolic flux redirection. Therefore, we constructed another *E. coli* strain, “PHB con+crRNA”, which expresses phbCAB constitutively with its original promoter within the operon and expresses crRNA through CcaS/CcaA system (Figure 14). ![](https://static.igem.org/mediawiki/2018/f/fd/T--SDU-CHINA--R1.png) Figure 14. Gene circuit of “PHB con+crRNA” Furthermore, the expression of crRNA was turned on at different periods of PHB fermentation. To be more specific, “PHB con+crRNA 5h/13.5h” expressed crRNA at 5h (exponential phase) and 13.5h (stationary phase) respectively by switching from red light illumination into green light. And besides, “PHB con+crRNA Red” was illuminated with red light all the time while “PHB con+crRNA Green” expressed crRNA throughout the fermentation (lag phase). ![](https://static.igem.org/mediawiki/2018/b/b5/T--SDU-CHINA--R2.png) ![](https://static.igem.org/mediawiki/2018/c/c9/T--SDU-CHINA--R3.png) ![](https://static.igem.org/mediawiki/2018/c/c3/T--SDU-CHINA--R4.png) Figure 15. Cell growth curve and PHB production of strain “PHB con+crRNA”. In "PHB con+crRNA Green/5h/13.5h", the expression of crRNA is activated at the beginning (0h), 5h and 13.5h respectively. By contrast, "PHB con+crRNA Red" is incubated under red light all the time to suppress crRNA expression. (a) the cell growth curve during the PHB fermentation. (b) the ratio between PHB and cell drought weight. (c) total amount of PHB produced during the fermentation. As we can see, despite “PHB con+crRNA Red” grew slightly better than the other, there was no significant difference between “PHB con+crRNA Green/5h/13.5h” (Figure 15. a), which meant that unlike the results shown on crRNA characterization, here, crRNA doesn’t inhibit cell grow. Based on the former PHB fermentation result, this was probably because the PHB synthesized by *E. coli* also has a positive effect on cell growth which offsets the detrimental impact of crRNA. And since crRNA was just expressed at a leaky level, “PHB con+crRNA Red” seems to grow slightly better than the other groups. With respect to PHB production, “PHB con+crRNA Red” with leaky expression of crRNA produced less PHB showing that the inhibition of *gltA* gene by crRNA improved PHB production dramatically (Figure 15. b c). And more importantly, the amount of PHB within the cells is different among different “PHB con+crRNA” group (Figure 15. b). That’s to say, the expressing crRNA at different periods of fermentation might lead to the variation of PHB content. In addition, “PHB con+crRNA 5h” tended to produce more PHB as shown in the data (Figure 15. b c) in comparison to “PHB con+crRNA Green/13.5h”. Thus, we can express crRNA at exponential phase to improve PHB production while turning on crRNA at stationary phase would be a bad choice. In short, expressing crRNA can improve PHB synthesis at exponential phase would be the best choice of crRNA expression. After demonstrating the expression of phbCAB cluster and the effect of crRNA, we intend to combine light control PHB synthesis and *gltA* inhibition together for further demonstration of the possibility of our growth-production switching system in the future. Besides, we will also make use of the powerful technique, HPLC, to show the exact changes in metabolic flux after *gltA* is repressed by crRNA. References [1] Han, Tiyun, Quan Chen, and Haiyan Liu. "Engineered photoactivatable genetic switches based on the bacterium phage T7 RNA polymerase." ACS synthetic biology 6.2 (2016): 357-366. [2] Baumschlager, Armin, Stephanie K. Aoki, and Mustafa Khammash. "Dynamic blue light-inducible T7 RNA polymerases (Opto-T7RNAPs) for precise spatiotemporal gene expression control." ACS synthetic biology 6.11 (2017): 2157-2167. [3] Mitsuharu Nakajima, Stefano Ferri, Matthias Rögner and Koji Sode. "Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal." Scientific Reports 6(2016):37595. [4] Yizhao Chang, Tianyuan Su, Qingsheng Qi and Quanfeng Liang. ”Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system” Microb Cell Fact (2016) 15:195. [5] Wang Q, Yu H, Xia Y, et al. Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application[J]. Microbial cell factories, (2009), 8(1): 47.

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