iGEM 2023 — Team hk-skhlpss

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| 2022 | Progress | Venue |
| --- | --- | --- |
| December | Team formed for iGEM2023 | S.K.H. Li Ping Secondary School |
| 2023 | Progress | Venue |
| January | Gathered project ideas and background research | S.K.H. Li Ping Secondary School |
| March | iGEM2023 registration | S.K.H. Li Ping Secondary School |
| April | Designed wet lab protocol | S.K.H. Li Ping Secondary School |
| June | Started wet lab on DNA duplex performance investigation | S.K.H. Li Ping Secondary School |
| August 12 | Human Practice: iGEM 2023 HK-MACAU Symposium | The Chinese University of Hong Kong |
| August 19 | Human Practice: High School Synthetic Biology Workshop | The Chinese University of Hong Kong |
| August 25 | Human Practice: Joint School Science Competition Visit | Hong Kong Central Library |
| August 28 | Human Practice: Intra School Summer Workshop | S.K.H. Li Ping Secondary School |
| September to October | Human Practice: Intra School Synthetic Biology Promotion | S.K.H. Li Ping Secondary School |
| September | Cloning (Collaborated by HongKong-CUHK iGEM Team) | The Chinese University of Hong Kong |
| September 16 | Human Practice: Joint Team Primary School Workshop | SKH Chu Oi Primary School (Lei Muk Shue) |
| November | Giant Jamboree | Paris, France |

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# Biobrick Design

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![](https://static.igem.wiki/teams/4837/wiki/engineering-photo-1.png)

The design of our plasmid was illustrated in the graph on the left. Since the conjugated product in our project involves both enzymes and oligonucleotides, our team plan to synthesize enzymes used in this project. Hence, two basic parts were designed, including lignin peroxidase (LiP) [Part ID: [BBa\_K4837000](https://parts.igem.org/Part%3ABBa_K4837000)] and manganese peroxidase (MnP) [Part ID: [BBa\_K4837001](https://parts.igem.org/Part%3ABBa_K4837001)], which were engineered using pSBIK3 vector. These sequences were optimized for *E.coli* expression.

The promoter used in these two basic parts was arabinose promoter. Purification tag and fluorescent tag were also included for better detection of the optimized enzyme synthesized.

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# Optimization of DNA duplex performance

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Different lengths of DNA duplex containing same number of nucleotides were designed in accordance with complementary base pairing principle. Each oligonucleotide pair (50nM, 100μL each) sponsored by IDT Technologies were mixed and placed in the Dynamica C-Master Gradient thermal cycler. Starting at 95ºC, the temperature was then dropped 1ºC per minute until room temperature was reached. Polyacrylamide gel electrophoresis (PAGE) was then conducted to check for the stability of each DNA duplex.

| Oligo | Sequence | Size (nucleotide) |
| --- | --- | --- |
| Oligo 1 | TGCCGGGTATAGCGCTTAGCATCCCGACACCTCTACCTACGCGGCCCAAACCAACAGGGC | 60 |
| Oligo 2 | GCCCTGTTGGTTTGGGCCGCGTAGGTAGAGGTGTCGGGATGCTAAGCGCTATACCCGGCA | 60 |
| Oligo 3 | GGTTCCTCTTGCTTACGGTCGGCGGACTAGGCCGTACCTACCATC | 45 |
| Oligo 4 | GATGGTAGGTACGGCCTAGTCCGCCGACCGTAAGCAAGAGGAACC | 45 |
| Oligo 5 | GCTGTTATGAAATAGCGGGAGGTTAGACGTCAGGT | 35 |
| Oligo 6 | ACCTGACGTCTAACCTCCCGCTATTTCATAACAGC | 35 |
| Oligo7 | TCGTCATAGGCCGCACGAGTTAGAT | 25 |
| Oligo 8 | ATCTAACTCGTGCGGCCTATGACGA | 25 |
| Oligo 9 | TCATCACACTTGGAT | 15 |
| Oligo 10 | ATCCAAGTGTGATGA | 15 |

###### The composition of each lane is documented below:

##### 1st Gel

| Lane | Composition |
| --- | --- |
| 1 | Oligo 1 |
| 2 | Oligo 2 |
| 3 | Oligo 1 + Oligo 2 |
| 4 | Oligo 3 |
| 5 | Oligo 4 |
| 6 | Oligo 3 + Oligo 4 |
| 7 | Oligo 5 |
| 8 | Oligo 6 |
| 9 | Oligo 5 + Oligo 6 |

##### 2nd Gel

| Lane | Composition |
| --- | --- |
| 1 | Oligo 7 |
| 2 | Oligo 8 |
| 3 | Oligo 7 + Oligo 8 |
| 4 | Oligo 9 |
| 5 | Oligo 10 |
| 6 | Oligo 9 + Oligo 10 |

# Preparation of oligonucleotide and enzymes

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After selection of the most stable DNA duplex, the sequences were ordered from our sponsor Integrated DNA Technologies. The sequences we ordered were undergone azide modification with an NHS Ester functional group to attach an azide moiety at the 5' of the oligos.

The enzymes, including lignin peroxidase (LiP) and manganese peroxidase (MnP) were engineered using pSBIK3 vector.

# Conjugation of DNA oligo and enzyme

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* 100 μg of enzyme was incubated with Dibenzocyclooctyne (DBCO) (purchased from Sigma-Aldrich) in PBS for 2 h at room temperature. DBCO was then removed with centrifugal filter.
* The DBCO-functionalized enzyme MnP and LiP were reacted with azide-modified oligonucleotide MnP\_O1 and LiP\_O2 respectively.
* Conjugation was conducted at 4°C for 16 hours. The unreacted oligonucleotides were removed with centrifugal filter.

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# Optimization of DNA duplex performance

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The below gel photos showed the result of optimization of DNA duplex performance using 8% DNA Polyacrylamide Gel Electrophoresis (PAGE).

[![](https://static.igem.wiki/teams/4837/wiki/photo-1-results-gel1.jpeg)](https://static.igem.wiki/teams/4837/wiki/photo-1-results-gel1.jpeg)

[![](https://static.igem.wiki/teams/4837/wiki/photo-2-results-gel2.jpeg)](https://static.igem.wiki/teams/4837/wiki/photo-2-results-gel2.jpeg)

From the result, it is clearly shown that bands appeared in each DNA duplex. Although multiple bands are shown in the lane containing DNA duplex for oligo 9+10, the band at the bottom below the corresponding DNA ladder is expected to be the DNA duplex product. It is suggested that the band intensity in the lane containing oligo 1+2, are the highest compared to lanes containing
oligo 3+4, oligo 5+6,
oligo 7+8 and oligo 9+10 respectively. It is suggested that the mixture of oligo 1 and oligo 2 possess the highest stability, compared to different DNA duplex, which is selected to be used in the enzyme conjugation.

No band was observed for oligo 5 and oligo 6 in 1st gel and oligo 9 and oligo 10 in 2nd gel. The relative high concentration of the DNA ladder resulted in a rather short exposure time for the gel. These bands are expected to appear when higher concentration of samples is prepared.

# Conjugation of DNA oligo and enzyme

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Conjugation of DNA oligo and enzyme
Conjugation was successful using 50uM of DBCO, with MnP to oligo ratio at 2:1. With the oligo dye, the lower band here is expected to be the oligos without conjugation with MnP, while this band is expected to be the oligos with conjugation.
No bands were found even 50uM DBCO used. What we learn is that it may be due to wrong filter size used in purification. The size of LiP is about 47kDa, while the filter size is 50kDa.

[![](https://static.igem.wiki/teams/4837/wiki/result-photo3.jpeg)](https://static.igem.wiki/teams/4837/wiki/result-photo3.jpeg)

[![](https://static.igem.wiki/teams/4837/wiki/result-photo4.jpeg)](https://static.igem.wiki/teams/4837/wiki/result-photo4.jpeg)

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