iGEM 2018 — Team HUBU-Wuhan

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# Team:HUBU-Wuhan/Notebook

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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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# Team:HUBU-Wuhan/Experiments

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## Protocol

# 1. Primer design:

    15bp homologous arm（Forward:ATCCTCAGTGTCAGC; Reverse:GCCCTTGCTCACCAT）+ DNA sequences.

![](https://static.igem.org/mediawiki/2018/8/84/T--HUBU-Wuhan--ptc1.png)

# 2. Amplification of target gene:

    Take the total DNA as a template, using designed primers to obtain target fragments with promoters.

![](https://static.igem.org/mediawiki/2018/0/0e/T--HUBU-Wuhan--ptc2.png)

# 3. Construction of recombinant plasmids:

    T5 Enzyme Assisted Cloning.

        1) Prepare reactive system (5 μL).
            **F/V**: molar ratio of 3:1
            **T5 enzyme**: 0.5 μL
            **Buffer**: 0.5 μL
            **ddH2O**: complemented to 5 μL
    All regents were mixed and reacted on the ice for 5 minutes.

    2) Add 100 μL DH5α competent cell and reacted on the ice 30 min.

    3) 42℃, heat shock 45 s, standing in 2-3 min.

    4) Add 100 μL NZY to recovery, culture at 37℃ with 250 rpm for 1 h.
            {NZY medium: 1% NZ-Amine, 0.5% yeast extract, 0.5% NaCl, (autoclaving), 12.5 mmol/L MgCl2·6H2O, 12.5 mmol/L MgSO4·7H2O, 20 mmol/L glucose (filtration sterilization)}.

    5) Plate cells on LB agar plates with Spe(20 mg/mL ).

# 4. Screening *E.coli* recombinant strain:

    10 μL aseptic water was taken from the super-clean workbench and 8 single colonies were taken from each sample in a PCR tube. The samples were dissolved in 10 μL aseptic water as templates.

![](https://static.igem.org/mediawiki/2018/e/e8/T--HUBU-Wuhan--ptc3.png)

# 5. Sequencing:

    Sending the sample to sequencing. Analysing the sequencing result with Snapgene. If the sequencing result of plasmid was correct, preserve the strain.

# 6. Electrotransformation:

    1) Precool the cuvettes and prepare competent cell in the ice.
    2) Mix plasmid (0.5-10 ng) and 50 μL competent cells in the cooling cuvette.
    3) Carefully dry the water on the outside of cuvette, and then insert the cuvette into the electroporator and press the start button.
    4) Add 1 mL liquid medium (RM: rich media) to recovery bacteria and incubate at 30℃for 6-12 h RM: 20.0 g/L, glucose; 10.0 g/L, yeast extract; 2.0 g/L, KH2PO4.
    5) Plate cells on LB agar plates with special antibiotic marker. LB: 10.0 g/L tryptone, 5.0 g/L yeast extract, and 10.0 g/L NaCl.

# 7. FACS:

    Flow cytometer was used to detect fluorescence. Record the data.

![](https://static.igem.org/mediawiki/2018/d/df/T--HUBU-Wuhan--cld.png)
![](https://static.igem.org/mediawiki/2018/4/44/T--HUBU-Wuhan--ptc7.JPG)

# 8. Double Digestion:

    1) Add 5ul 10X FlyCut buffer to PCR tube.
    2) Add an amount containing 1ug of DNA sample. If DNA concentration is <62.5ng/uL, add 16ul.
    3) Add 0.5ul Endonuclease each.
    4) For total volume<50ul, add ddH2O to make up 50ul.
    5) Incubate at 37°C for 30min.

# 9. Ligation:

    1) Measure concentration of insert and vector.
    2) Add at least 20ng of vector.
    3) Add insert in 1:3 vector to insert ratio.
    4) Add 2uL of 5X T4 Ligase Buffer.
    5) Add 0.5uL of T4 Ligase.
    6) Top up till 10uL with ddH2O.
    7) Incubate at 4℃ temperature for 18 hours.

# 10. Transformation:

    1) Thaw Competent Cells on ice.
    2) Add 10ul ligated product or plasmid into Competent Cell and quickly place cell back on ice, incubating for 30mins.
    3) Conduct heat shock at 42°C for 45s.
    4) Chill cell on ice for 3min.
    5) Add 100uL NZY to cell.
    6) Incubate at 37°C for 1h.
    7) Spread 200uL of culture on LB antibiotic agar plate.
    8) Culture overnight at 37°C.
    9) Store plate at 4°C.

# 11. Isobutanol production measurement(HPLC):

    1) Inoculate strains for overnight culture in 5mL RMG5 in Culture Flask at 30°C.
    2) Measure the OD600 of the overnight culture with 200uL of sample.
    3) Dilute OD600 to 0.001 in 4mL RMG5 media in Deep-well Multiwell Plate and incubate in 30°C.
    4) Take samples after 24 hours by filtration sterilization.
    5) Make HPLC measurement and sort data.

![](https://static.igem.org/mediawiki/2018/1/18/T--HUBU-Wuhan--ptc11.JPG)

# 12. Alkaline hydrolysis:

    1) Mix 12% NaOH solution and material(the waste carton) in ratio 15:1 (mL:g).
    2) It’s placed in the constant temperature shaker ,and it’s treated under 55°and the speed of 150r/min for 1.5h.

# 13. Acid hydrolysis:

    1) Mix 3% H2SO4 solution and material(the waste carton) in ratio 15:1 (mL:g).
    2) It’s placed in the constant temperature shaker ,and it’s treated under 55°and the speed of 150r/min for 1.5h.
    3) Adjust pH to 4.8 by adding NaOH.

**References:**
    [1]Jingjing Zhang,etal.
Journal of Anhui Agri Sci,2009,37(35):17312-17314.

    [2]Duan Yuan,etal.
[J].Renewable Energy Sources,2011,29(3):30-34.

    [3]Xiao Lingping,Tang Yong,Deng Lihong.Enzymatic hydrolysis of waste corrugated cellulose and changes in fiber structure during enzymatic hydrolysis[J].Forestry Chemicals and Industry,2010,30(02):83-88.

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# Team:HUBU-Wuhan/Results

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## Results

# PART 1: Conversion of Waste Paper Cartons into Fermentable Sugars

    The primary ingredient of paper cartons is cellulose (ca 63 %). Pretreatment using H2SO4 and the subsequent enzymatic hydrolysis will break down the recalcitrant structure of cartons to release the fermentable mono-sugars. We compared the glucose yield from different enzymes and different amounts enzyme. In combination with the results of bacterial culture, we finally selected two times the amount of solid enzyme as the final scheme of the experiment.

![](https://static.igem.org/mediawiki/2018/6/69/T--HUBU-Wuhan--rst1.png)

**Figure 1. Glucose yield of two kinds of enzyme.**

    The highest glucose yield is that 12.7 g glucose can be extracted from 3 g waste paper cartons , which can be directly used as the carbon source for fermentation to produce cellulosic ethanol without any centrifugation. The glucose we produced an successfully cultured the ZM4 and produced ethanol.

![](https://static.igem.org/mediawiki/2018/3/3e/T--HUBU-Wuhan--rst2.png)

**Figure 2.The Glucose and Ethanol yield by Fermentation.**

# PART 2: Construction of Isobutanol-production Module

    Isobutanol is toxic to cells. So we measured the isobutanol tolerance of *Zymomonas mobilis*. The following results showd that it can tolerate up to 1.5% concentration of isobutanol.

![](https://static.igem.org/mediawiki/2018/4/42/T--HUBU-Wuhan--rst3.png)

**Figure 3. Isobutanol toxicity.**

    We transformed the expression plasmid pEZ15A which contains the fragment ‘Ptet-kdcA’ to *Zymomonas mobilis*. The gene kdcA is induced by the inducible promoter, Ptet. We measured the strain’s growth and isobutanol production under three different concentration of tetracycline. With the increase of concentration of Tet, the growth rate and the sugar utilization rate will also be affected, they are in a coupling state. As the Tet increased, the growth of the bacteria would be Inhibited, but the yield of isobutanol increased and finally reached around 100 mg/L.

![](https://static.igem.org/mediawiki/2018/4/41/T--HUBU-Wuhan--rst4.1.png)

**Figure 4. Isobutanol production gene.**

    Different candidate genes for isobutanol were selected and tested to build an isobutanol production module to maximize carbon flux into isobutanol production. We constructed following 5 kinds of pEZ15A plasmids which contain different isobutanol production module. Up to now, we have transformed pEZ15A-Peno-kdcA into ZM4 strain and 8b strain and pEZ15A-Pgap-kdcA into 8b strain. We measured their isobutanol production. The isobutanol production of ZM4 strain with pEZ15A-Peno-kdcA reached 121 mg/L and the isobutanol production of 8b strain with pEZ15A-Peno-kdcA and pEZ15A-Pgap-kdcA reached 147 mg/L and 97 mg/L respectively. As control, the ZM4 and 8b strain transformed with blank pEZ15A plasmid all can not produce isobutanol.

![](https://static.igem.org/mediawiki/2018/9/9c/T--HUBU-Wuhan--rstt1.png)

**Table 1. five kinds of isobutanol production plasmids.**

![](https://static.igem.org/mediawiki/2018/c/c6/T--HUBU-Wuhan--rst5.png)

**Figure 5. Isobutanol production of different strains.**

    Next we will transform the other isobutanol production module into the ZM4 strain and 8b strain and transform these modules to other microorganisms to test their efficiency and compatibility.

# PART 3: Promoter Optimization

**① regulable promoters**

    To test the change of our promoters’ strength, we constructed a dual-reporter system. A dual-reporter system was developed to quantify the strength of these promoters, which contains mCherry reporter gene driven by constitutive PlacUV5 promoter for calibration, and eGFP reporter gene to quantify the candidate promoter. In mid-September, 3 candidates were selected due to their outstanding increase of strength, one of which is up-regulated in stationary phase(P0038), the rest of which are ethanol inducible(P0435,P0405).

![](https://static.igem.org/mediawiki/2018/6/6a/T--HUBU-Wuhan--rst6.jpg)

**Figure 6. Strength change of P0405 during growth without ethanol induce.**

![](https://static.igem.org/mediawiki/2018/9/96/T--HUBU-Wuhan--rst7.jpg)

**Figure 7. Strength change of P0435 during growth without ethanol induce.**

![](https://static.igem.org/mediawiki/2018/e/ea/T--HUBU-Wuhan--rst8.jpg)

**Figure 8. Strength change of P0038 during growth without ethanol induce.**

    Compared with other candidates, these 3 promoters showed Superior performance.The promoter of ZMO0405, ZMO0435 and ZMO0038 has 2-fold change comparing exponential phase and stationary phase. These three promoters should pay more attention and repeat the experiments to confirm the potential regulatory mechanisms.

![](https://static.igem.org/mediawiki/2018/6/69/T--HUBU-Wuhan--rst9.jpg)

**Figure 9. (a)Fluorescence intensity of regulable promoters without ethanol induce in *Z.mobilis*. (b)EGFP/mCherry of regulable promoters without ethanol induce in *Z.mobilis*.(20180908-1st)**

![](https://static.igem.org/mediawiki/2018/7/75/T--HUBU-Wuhan--rstt2.jpg)

**Table 2. The FACS result of regulable promoters.(20180908-1st,initial OD600 nm is 0.1.)**

![](https://static.igem.org/mediawiki/2018/f/f8/T--HUBU-Wuhan--rst10.jpg)

**Figure 10. (a)Fluorescence intensity of regulable promoters without ethanol induce in *Z.mobilis*. (b)EGFP/mCherry of regulable promoters without ethanol induce in *Z.mobilis*.(20180908-2nd)**

![](https://static.igem.org/mediawiki/2018/e/ee/T--HUBU-Wuhan--rstt3.jpg)

**Table 3. The FACS result of regulable promoters.(20180908-2nd,initial OD600 nm is 0.1.)**

![](https://static.igem.org/mediawiki/2018/a/a7/T--HUBU-Wuhan--rst11.jpg)

**Figure 11. (a)Fluorescence intensity of regulable promoters with 5% ethanol induce in *Z.mobilis*. (b)EGFP/mCherry of regulable promoters with 5% ethanol induce in *Z.mobilis*.(20180914-2nd)**

![](https://static.igem.org/mediawiki/2018/9/91/T--HUBU-Wuhan--rst12.jpg)

**Figure 12. (a)Fluorescence intensity of regulable promoters with 5% ethanol induce in *Z.mobilis*. (b)EGFP/mCherry of regulable promoters with 5% ethanol induce in *Z.mobilis*.(20180918-3rd)**

![](https://static.igem.org/mediawiki/2018/d/d0/T--HUBU-Wuhan--rst13.jpg)

**Figure 13. (a)Fluorescence intensity of regulable promoters with 5% ethanol-stress in *Z.mobilis*. (b)EGFP/mCherry of regulable promoters with 5% ethano-stress in *Z.mobilis*.(20180918-1st)**

**More raw data click >>**
    →[9.8\_no\_treatment](https://static.igem.org/mediawiki/2018/a/a7/T--HUBU-Wuhan--9.8_no_treatment.xlsx)
    →[9.10\_EtOH-stress\_3\_promoter](https://static.igem.org/mediawiki/2018/7/76/T--HUBU-Wuhan--9.10_EtOH-stress_3_promoter.xlsx)
    →[9.11\_EtOH\_induce\_all](https://static.igem.org/mediawiki/2018/7/75/T--HUBU-Wuhan--9.11_EtOH_induce_all.xlsx)
    →[9.14\_EtOH\_induce\_all](https://static.igem.org/mediawiki/2018/3/3f/T--HUBU-Wuhan--9.14_EtOH_induce_all.xlsx)
    →[9.14\_no\_treatment](https://static.igem.org/mediawiki/2018/5/51/T--HUBU-Wuhan--9.14_no_treatment.xlsx)
    →[9.19\_EtOH\_induce\_all](https://static.igem.org/mediawiki/2018/6/68/T--HUBU-Wuhan--9.19_EtOH_induce_all.xlsx)

**② mutant Pgap**

    Pgap random mutagenesis by error-prone PCR.
    Sorting stronger mutations in *E. coli* using FACS.
    We screened a total of 42 mutants (12 mutant types) by constructing a mutant library, Cell sorting,and sequencing.

![](https://static.igem.org/mediawiki/2018/d/d3/T--HUBU-Wuhan--rst14.png)

**Fig.14 All mutants information.**

    Then we test the expression of mutants in *E. coli* by FACS.
    Experimental conditions:4h,8h, 5 mL, 200 rpm，Pgap Mutants in *E. coli*.

![](https://static.igem.org/mediawiki/2018/3/3c/T--HUBU-Wuhan--rst15.png)

**Fig.15 The first FACS, the expression of mutant strains after 4 hours[Log phase]of culture.**

    After 4 hours, the growth of the mutant strain entered the log phase. Compared with WT, we can see that most of the mutants have enhanced GFP fluorescence. Initially, this means that our mutation is effective and the expression of Pgap is improved.

![](https://static.igem.org/mediawiki/2018/6/67/T--HUBU-Wuhan--rst16.png)

**Fig.16 The first FACS, the expression of mutant strains after 8 hours[stable phase] of culture.**

    After 8 hours, the growth of the mutant strain entered the stable phase. Compared with WT, we can see that the GFP fluorescence of most mutants is still enhanced, and the expression of Pgap is improved.

![](https://static.igem.org/mediawiki/2018/a/a2/T--HUBU-Wuhan--rst17.png)

**Fig.17 The first FACS, Fluorescence intensity of Pgap mutants in *E. coli*.**

    From the statistical data, Compared with WT, 3S-2, 3S-11, 3S-14, 4S-P2-4, 4S-P2-6, 4S-P1-18, 4S-P1-1, 3S-P1-4 GFP Fluorescence intensity is significantly improved in both log phase and stable phase.

![](https://static.igem.org/mediawiki/2018/b/bd/T--HUBU-Wuhan--rst18.png)

**Fig.18 The first FACS, EGFP[driven by candidate Pgap mutants]/mCherry [internal control]of Pgap mutants in *E. coli*.**

    However, we used the ratio data of GFP and mcheery which better express the expression of Pgap mutants. The ratio of 4s-P1-1 and 3S-P1-4 was lower than WT, which means that the two mutants Expression has not been enhanced.
    In order to achieve repeatability of the experiment, we repeated an experiment and performed a second FACS.
    Experimental conditions:4h, 8h, 3 mL, 200 rpm ，12 Pgap Mutants in *E. coli*.

![](https://static.igem.org/mediawiki/2018/b/b2/T--HUBU-Wuhan--rst19.png)

**Fig.19 The second FACS, the expression of mutant strains after 4 hours[Log phase]of culture.**

    In the second experiment, after 4 hours, the growth of the mutant strain entered the log phase. Compared with WT, we can also see that most of the mutants have enhanced GFP fluorescence. Initially, this means that our mutation is effective and the expression of Pgap is improved.

![](https://static.igem.org/mediawiki/2018/f/f7/T--HUBU-Wuhan--rst20.png)

**Fig.20 The second FACS, the expression of mutant strains after 8 hours[stable phase] of culture.**

    In the second experiment, after 8 hours, the growth of the mutant strain entered the stable phase. Compared with WT, we can see that the GFP fluorescence of most mutants is still enhanced, and the expression of Pgap is improved.

![](https://static.igem.org/mediawiki/2018/1/17/T--HUBU-Wuhan--rst21.png)

**Fig.21 The second FACS, Fluorescence intensity of Pgap mutants in *E. coli*.**

    In the second experiment, compared with WT, 3S-2, 3S-11, 3S-14, 4S-P2-4, 4S-P2-6, 4S-P1-18, 3S-P1-4 GFP Fluorescence intensity is significantly improved in both log phase and stable phase.

![](https://static.igem.org/mediawiki/2018/3/3b/T--HUBU-Wuhan--rst22.png)

**Fig.22 The second FACS, EGFP[driven by candidate Pgap mutants]/mCherry [internal control]of Pgap mutants in *E. coli*.**

    Same as the first time,t he ratio of 3S-P1-4 was lower than WT, which means that the mutant ‘s Expression has not been enhanced. From the data of these two times, the expression of mutants 3S-2, 3S-11, 3S-14, 4S-P2-4, 4S-P2-6, 4S-P1-18 was enhanced.

![](https://static.igem.org/mediawiki/2018/4/46/T--HUBU-Wuhan--rst23.png)

**Fig.23 Correlation between two replicates.**

    The correlation of the two replicates is relatively high with an R2 0.9366, which means the result is reliable. Additionally, we obtained 6 mutants which have more than 1.5-fold changes compared with wild type Pgap.

![](https://static.igem.org/mediawiki/2018/3/3a/T--HUBU-Wuhan--rst24.png)

**Fig.24 Fluorescence intensity of EGFP.**

![](https://static.igem.org/mediawiki/2018/f/f4/T--HUBU-Wuhan--rst25.png)

**Fig.25 Fold change of mutants.**