iGEM 2018 — Team HKJS_S

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# Team:HKJS S/Notebook

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TEAM

[Team Members](https://2018.igem.org/Team%3AHKJS_S/Team)
[Collaborations](https://2018.igem.org/Team%3AHKJS_S/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AHKJS_S/Description)
[Design](https://2018.igem.org/Team%3AHKJS_S/Design)
[Experiments](https://2018.igem.org/Team%3AHKJS_S/Experiments)
[Notebook](https://2018.igem.org/Team%3AHKJS_S/Notebook)
[InterLab](https://2018.igem.org/Team%3AHKJS_S/InterLab)
[Model](https://2018.igem.org/Team%3AHKJS_S/Model)
[Results](https://2018.igem.org/Team%3AHKJS_S/Results)
[Demonstrate](https://2018.igem.org/Team%3AHKJS_S/Demonstrate)
[Improve](https://2018.igem.org/Team%3AHKJS_S/Improve)
[Attributions](https://2018.igem.org/Team%3AHKJS_S/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AHKJS_S/Parts)
[Basic Parts](https://2018.igem.org/Team%3AHKJS_S/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AHKJS_S/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AHKJS_S/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AHKJS_S/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AHKJS_S/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AHKJS_S/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AHKJS_S/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AHKJS_S/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AHKJS_S/Hardware)
[Measurement](https://2018.igem.org/Team%3AHKJS_S/Measurement)
[Model](https://2018.igem.org/Team%3AHKJS_S/Model)
[Plant](https://2018.igem.org/Team%3AHKJS_S/Plant)
[Software](https://2018.igem.org/Team%3AHKJS_S/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=HKJS_S)

**Notebook**

[Show all][Hide all]

Week 1 29-6-18[Show]

Completed calibration 1&2. Data is not accurate, possibly due to pipetting errors.
[Hide]

Week 2 3-6 July[Show]

Repeated calibration 1&2, started calibration 3. Data is linearized
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Week 3 9-13 July [Show]

Repeated calibration 1-3
[Hide]

Week 4 16-20 July [Show]

Started transformation for cell measurement protocol. No cells grew, possibly due to low competency of E. coli.
[Hide]

Week 5 23-27 July [Show]

Repeated calibration 1-3, took the average of all data. Prepared cells using method suggested in The Inoue Method for Preparation and Transformation of Competent E. coli. Completed fluorescence and absorbance cell measurement, and Colony Forming Units. Handed in data.
[Hide]

Week 6 31July to 3Aug [Show]

Assembled DNA fragments using Gibson assembly and ligation.
Fragment 1+ Vector+ Fragment 5, fragment 2+3+4
The final product is named Fp1.
Performed gel electrophoresis, but the fragment is stuck in the well.
![](https://static.igem.org/mediawiki/2018/7/7f/T--HKJS_S--Log--731_3.jpg)

Week 7 6-10 Aug [Show]

Transformed Fp1 into NEB5α, picked single colonies and grew overnight in LB. Performed plasmid purification and gel ‘electrophoresis, band size is smaller than expected (expected size is about 11kb). Performed restriction enzyme digestion.
[Hide]

Week 8 13-17 Aug [Show]

Assembled DNA fragments using Gibson assembly and ligation.
Transformed stock of Fp1 to NEB5α again. Picked colonies from previous transformation and incubated overnight. Performed plasmid purification and gel electrophoresis. Band size smaller than expected. Assemble the fragments again using Gibson Assembly, with Echo liquid handler. The product is named Fp2. Transformed Fp2 into NEB5α, picked colonies and plasmid purified.
![](https://static.igem.org/mediawiki/2018/e/e7/T--HKJS_S--Log_1.png)

Week 9 20-24 Aug [Show]

Performed restriction enzyme cut and gel electrophoresis for the transformation of Fp2. Band size smaller than expected. Tested transformation efficiency for BL21 and NEB5α. Found out that pETBlue-2 does not seem to enter the BL21 strain. Prepared ultra competent cells using the method suggested in The Inoue Method for Preparation and Transformation of Competent E. coli.
![](https://static.igem.org/mediawiki/2018/f/f9/T--HKJS_S--Log_3.png)

Week 10 27-31 Aug [Show]

Transformed Fp1 and Fp2. Incubated overnight and plasmid purified 30 samples. Conducted electrophoresis and found that Fp1-7, Fp1-16 and Fp1-A have similar band size as expected (about 11kb). Further performed restriction enzyme double cut and electrophoresis. All the above plasmids are in optimal size.
Picked colonies for MHETase and ran colony PCR check. No band at all. Ran colony PCR check again, still no expected band.
![](https://static.igem.org/mediawiki/2018/3/36/T--HKJS_S--Log_5.png)
The sequencing results of PETase showed that there was no insert in the plasmid. Therefore, we performed PCR, restriction enzyme digestion and ligation for PETase again. Transformed into E. coli and performed plasmid purification. Restriction enzyme digest the plasmid purified products to check insert size. The results showed that there was no insert.
[Hide]

Week 11-14 3-28 Sept [Show]

Transformed Fp1 and Fp2 into NEB5α. Picked single colonies and incubated overnight. Performed plasmid purification, restriction enzyme digestion and gel electrophoresis. Some plasmid sizes are as expected.
Gel Electrophoresis photos taken that week. Asseen, some plasmid sizes are as expected
e.g. Gel photo located second from the left in the bottom row, the plasmid size is as expected.

![](https://static.igem.org/mediawiki/2018/1/18/T--HKJS_S--Log_6.png)![](https://static.igem.org/mediawiki/2018/e/e6/T--HKJS_S--Log_7.png)

![](https://static.igem.org/mediawiki/2018/7/7d/T--HKJS_S--equipments.png)
Successful samples   Materials used throughout the project
After restriction enzyme digestion and ligation to pETBlue-2, the product was transformed to E. coli. Subsequent plasmid purification was carried out. Samples were sent to HKUST for SDS-PAGE to check protein size. The size of protein is confirmed.
![](https://static.igem.org/mediawiki/2018/7/72/T--HKJS_S--colab--gel.png)
[Hide]

Week 15-16 1-9 Oct [Show]

Performed electrophoresis for the samples with expected sizes. Performed restriction enzyme digestion and ligated the samples to pSB1C3.Transformed the plasmid into NEB5α and performed plasmid purification. Sent the products to the iGEM headquarters.
[Hide]

Week 17 8-17 Oct [Show]

Discussions on the ideal environment for the gene to be expressed. Measured optical density for the modelling of the project.
[Hide]

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# Team:HKJS S/Experiments

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TEAM

[Team Members](https://2018.igem.org/Team%3AHKJS_S/Team)
[Collaborations](https://2018.igem.org/Team%3AHKJS_S/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AHKJS_S/Description)
[Design](https://2018.igem.org/Team%3AHKJS_S/Design)
[Experiments](https://2018.igem.org/Team%3AHKJS_S/Experiments)
[Notebook](https://2018.igem.org/Team%3AHKJS_S/Notebook)
[InterLab](https://2018.igem.org/Team%3AHKJS_S/InterLab)
[Model](https://2018.igem.org/Team%3AHKJS_S/Model)
[Results](https://2018.igem.org/Team%3AHKJS_S/Results)
[Demonstrate](https://2018.igem.org/Team%3AHKJS_S/Demonstrate)
[Improve](https://2018.igem.org/Team%3AHKJS_S/Improve)
[Attributions](https://2018.igem.org/Team%3AHKJS_S/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AHKJS_S/Parts)
[Basic Parts](https://2018.igem.org/Team%3AHKJS_S/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AHKJS_S/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AHKJS_S/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AHKJS_S/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AHKJS_S/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AHKJS_S/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AHKJS_S/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AHKJS_S/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AHKJS_S/Hardware)
[Measurement](https://2018.igem.org/Team%3AHKJS_S/Measurement)
[Model](https://2018.igem.org/Team%3AHKJS_S/Model)
[Plant](https://2018.igem.org/Team%3AHKJS_S/Plant)
[Software](https://2018.igem.org/Team%3AHKJS_S/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=HKJS_S)

**Experiment**

Protocols[Show all]

Inoue Method for Preparation of “Ultra-competent” Cells[Show]

1. Prepare Inoue transformation buffer
2. Streak out bacteria from freezer stock on LB-agar plate and grow overnight at 37°C
3. Pick a single colony and transfer to 25 mL of LB media in a 250 mL flask
4. Incubate culture for 6 – 8 hours at 37°C with vigorous shaking
5. At ~6 pm, inoculate 3 1-liter flasks, each with 250 mL of LB media
     a. 1st flask = 10 mL of starter culture
     b. 2nd flask = 4 mL of starter culture
     c. 3rd flask = 2 mL of starter culture
6. Incubate 3 flasks overnight at 18°C
7. The following morning, take OD600 of the three flasks.
8. Monitor the flasks until one flask reaches OD600 = 0.55
9. Transfer this flask to an ice bath for 10 minutes and discard the other 2 cultures
10. Centrifuge cells at 2500g (~3500 rpm in sorvall) for 10 minutes at 4°C
11. Remove all media from samples. Use an aspirator if possible
12. Resuspend cells in 80 mL of ice-cold Inoue transformation buffer
13. Spin cells again at 2500g for 10 minutes at 4°C
14. Remove all buffer
15. Add 20 mL of ice-cold Inoue transformation buffer
16. Add 1.5 mL of DMSO
17. Mix the bacterial suspension by swirling and then store it on ice for 10 minutes
18. Dispense aliquots into eppendorf tubes and snap freeze in MeOH/dry ice
19. Store at -70°C

|  |  |
| --- | --- |
| **Inoue transformation buffer** | 1 liter |
| 55 mM MnCl2 | 10.88 g |
| 15 mM CaCl2 | 2.20 g |
| 250 mM KCl | 18.65 g |
| 10 mM PIPES (0.5 M, pH 6.7) | 20 mL |
| H2O | To 1 L |

PIPES
  - 15.1 g PIPES in 80 mL of H2O
  - Adjust to pH 6.7 with 5M KOH and then bring to 100 mL Sterilize by filtration
  - Store aliquots at -20°C

[Hide]

Gibson Assembly[Show]

1. Two or more gBlocks Gene Fragments are designed with 20–80 base overlaps with the adjacent gBlocks fragment sequences and the linearized plasmid. The plasmid can be linearized by restriction digest or PCR.
2. Linearized plasmid and fragments are combined in a tube with Gibson Assembly® Master Mix (New England Biolabs).
gBlocks® Gene Fragments and Plasmid Assembly

|  |  |  |
| --- | --- | --- |
| Total # of fragments including plasmid | 2–3 Fragments | 4–6 Fragments |
| Quantity1 | 0.02–0.50 pmole ea. | 0.05–5.0 pmole ea. |
| Gibson Assembly® Master Mix (2X) | 10 μL | 10 μL |
| Deionized H2O | Adjust to final 20 μL | Adjust to final 20 μL |
| Total volume | 20 μL | 20 μL |

1Use 50–100 ng of plasmid and a 2–3 fold excess of insert fragments. Table source: www.NEB.com/GibsonAssembly.
3. Incubate at 50oC for 1 hr.
4. The resulting completed plasmid is ready for transformation into bacteria, and analysis.

[Hide]

Experiment Methods[Show all]

Gene synthesis[Show]

The gene cluster which enables the carbon dioxide reduction is synthesised according to nif genes of Paenibacillus polymxya WLY 78, with amino acid substitutions of V70A and H195Q. The gene of interest is then silent mutated by editing the file to remove all restriction sites EcoRI, XbaI, SpeI, PstI, as to fulfil biobrick standards. Furthermore, biobrick prefix and suffix are added before and after the sequence. The resulted sequence is stated in the Supporting Information.
The gene of interest is synthesised with Integrated DNA Technologies (IDT) gblocks® Gene Fragments (gblocks®), a service that provides linear, double-stranded and sequenced DNA fragments [1]. As the length of gblocks® can only be up to 3000 bp [1], while nif genes are 10.9 kb in length, the ordering was separated into four parts, 2187, 2187, 2187 and 2389 in length respectively, with 50 bp overlapping region.

[1] Integrated DNA Technologies, “gBlocks® Gene Fragments,” [Online]. Available: http://sg.idtdna.com/pages/products/genes-and-gene-fragments/gblocks-gene-fragments. [Accessed 26 May 2018].
[Hide]

Gene resuspension[Show]

Upon the arrival of the gene fragments, the pellets are processed according to the instructions given by the manufacturer [2]. The pellets containing genes are first centrifugated 3-5 seconds with 3000x g to pellet the material to the bottom of the tube. Next, 20 µL TE is added to have a final concentration of 50 ng/µL, then vortexed and centrifuged briefly. The final processed genes are resuspended in in nuclease-free water.

[2] Integrated DNA Technologies, “gBlocks™ Gene Fragments Cloning Protocols,” [Online]. Available: http://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/gblocks-gene-fragments-protocols.pdf?sfvrsn=1c133407\_6. [Accessed 26 May 2018].
[Hide]

Assembly of gene cluster[Show]

The genes will be based on the assembly method proposed by Danial Gibson et. al. [3]. In the overlapping regions, a T5 exonuclease cleaves the 5' end of each fragment, resulting in overhangs, then the exonuclease is deactivated at 50ºC. This results in the annealing of overlapping fragments, hence a complete DNA cluster. Next, a DNA polymerase and ligase next fill in gaps and nicked parts in the gene of interest respectively.
Firstly, fragment 1 and 5 will be ligated to the linearized plasmid, then fragments 2, 3, 4 will be assembly by Gibson assembly. This results in two fragments containing the plasmid in one of them. Lastly, a Gibson assembly will be performed and can be directly used in transformation.

[3] D. G. Gibson, L. Young, R.-Y. Chuang, J. C. Venter, H. I. Clyde A and H. O. Smith, “Enzymatic assembly of DNA molecules up to several hundred kilobases.,” Nature Methods, vol. 6, pp. 343-345, 2009.
[Hide]

Transformation[Show]

The transformation will be done by Heat-shock method by the protocol provided by igem.

[Hide]

Selection[Show]

When transformed using pET-blue-2 as the plasmid backbone, a blue-white screen can be used to screen the colonies. The colony will be grown overnight in 2 mL of LB solution then the plasmid can be miniprepped, and subject to a gel electrophoresis screen.

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# Team:HKJS S/Results

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[HOME](https://2018.igem.org/Team%3AHKJS_S)

TEAM

[Team Members](https://2018.igem.org/Team%3AHKJS_S/Team)
[Collaborations](https://2018.igem.org/Team%3AHKJS_S/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AHKJS_S/Description)
[Design](https://2018.igem.org/Team%3AHKJS_S/Design)
[Experiments](https://2018.igem.org/Team%3AHKJS_S/Experiments)
[Notebook](https://2018.igem.org/Team%3AHKJS_S/Notebook)
[InterLab](https://2018.igem.org/Team%3AHKJS_S/InterLab)
[Model](https://2018.igem.org/Team%3AHKJS_S/Model)
[Results](https://2018.igem.org/Team%3AHKJS_S/Results)
[Demonstrate](https://2018.igem.org/Team%3AHKJS_S/Demonstrate)
[Improve](https://2018.igem.org/Team%3AHKJS_S/Improve)
[Attributions](https://2018.igem.org/Team%3AHKJS_S/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AHKJS_S/Parts)
[Basic Parts](https://2018.igem.org/Team%3AHKJS_S/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AHKJS_S/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AHKJS_S/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AHKJS_S/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AHKJS_S/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AHKJS_S/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AHKJS_S/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AHKJS_S/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AHKJS_S/Hardware)
[Measurement](https://2018.igem.org/Team%3AHKJS_S/Measurement)
[Model](https://2018.igem.org/Team%3AHKJS_S/Model)
[Plant](https://2018.igem.org/Team%3AHKJS_S/Plant)
[Software](https://2018.igem.org/Team%3AHKJS_S/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=HKJS_S)

Results

Results are derived after 5 times of Gibson Assembly, over 150 miniprep plasmid purification, nearly 100 gel electrophoresis and over 150 plates spreaded..

![](https://static.igem.org/mediawiki/2018/7/7d/T--HKJS_S--equipments.png)

**Equipment used**

Using The Inoue Method for Preparation and Transformation of Competent E. coli, all constructions are transformed into E. coli BL21 and Neb 5 alpha. Plasmid purification are done using the TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 and Monarch® Plasmid Miniprep Kit.pETBlue-2 is used for Blue-White screening.

# Nitrogenase

With reference to Fig. A below, it is observed that in the 3rd lane after an hour of 42V gel electrophoresis, a comparably larger fragment is constructed using Gibson Assembly.

![](https://static.igem.org/mediawiki/2018/6/62/T--HKJS_S--FigA.jpg)

**Fig. A**

All constructs in Fig. B are comparably larger products assembled by Gibson Assembly. The circular DNA on the 5th lane to the right is plasmid purified Gibson assembly product and the 4th lane from the right is its linearized form using double digestion EcoRI and PstI. This indicates that the product is Bio-Brick compatible. In addition, after several hours of gel electrophoresis with 42V, except for these two lanes mentioned above, all other bands migrate downwards to the 8kbp point after one hour. This concludes that among all constructs we have done, this product matches the most with our expectation.

![](https://static.igem.org/mediawiki/2018/5/50/T--HKJS_S--FigB.jpg)

**Fig. B**

These shows that we have successfully assembled the nif cluster using the Gibson Assembly.

# PET decomposition

As the high GC content make it difficult to complete PCR, we amplified the PETase and MHETase with PCR after codon optimization. For the reason that the band of MHETase is weak, we then try to perform gradient PCR using 6 different temperatures. After ligation and transformation, only PETase is successfully transformed. We express the protein and measured the size of it. With the help of HKUST team, the PETase is characterized using SDS-Page.

![](https://static.igem.org/mediawiki/2018/e/ec/T--HKJS_S--SDS.jpg)