iGEM 2019 — Team HK_SSC

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:HK SSC/Notebook

![]()

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3AHK_SSC)
[HOME](https://2019.igem.org/Team%3AHK_SSC)

TEAM

[Team Members](https://2019.igem.org/Team%3AHK_SSC/Team)
[Collaborations](https://2019.igem.org/Team%3AHK_SSC/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3AHK_SSC/Description)
[Design](https://2019.igem.org/Team%3AHK_SSC/Design)
[Experiments](https://2019.igem.org/Team%3AHK_SSC/Experiments)
[Model](https://2019.igem.org/Team%3AHK_SSC/Model)
[Notebook](https://2019.igem.org/Team%3AHK_SSC/Notebook)
[Contribution](https://2019.igem.org/Team%3AHK_SSC/Contribution)
[Results](https://2019.igem.org/Team%3AHK_SSC/Results)
[Demonstrate](https://2019.igem.org/Team%3AHK_SSC/Demonstrate)
[Improve](https://2019.igem.org/Team%3AHK_SSC/Improve)
[Attributions](https://2019.igem.org/Team%3AHK_SSC/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AHK_SSC/Parts)
[Basic Parts](https://2019.igem.org/Team%3AHK_SSC/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AHK_SSC/Composite_Part)

[SAFETY](https://2019.igem.org/Team%3AHK_SSC/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AHK_SSC/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AHK_SSC/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AHK_SSC/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AHK_SSC/Hardware)
[Measurement](https://2019.igem.org/Team%3AHK_SSC/Measurement)
[Model](https://2019.igem.org/Team%3AHK_SSC/Model)
[Plant](https://2019.igem.org/Team%3AHK_SSC/Plant)
[Software](https://2019.igem.org/Team%3AHK_SSC/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=HK_SSC)

17 July

Transformation of dcas9 plasmid 2019 kit plate 5 well 22n.

- kanamycin resistance plate

- to E. coli NEB5a

19 July

Picked 6 colonies

-grow overnight in LB

Plasmid purification (Takara)

-colony 1 & 2 did not grow

-run agarose gel for 100v 30 mins

-eluted DNA using 30ul elution buffer

-nanodrop the eluted DNA

22 July

Plasmid prep

Run gel for dCas9

Resuspend DNA

23 July

1. Assembling Fragment 1+2+3

-restriction endonuclease cut + ligation

NheI and BssSI-v2

1. Dcas9 preparation clean- Run gel NEB5α

Transformed produce to E.coli neb5

1. Cloned GFP to dcas9

-restriction endonuclease cut + ligation

EcorI NsiI

Transformed product to E.coli neb5

1. Transformed puc19 from stock (for cloning sgrna later)

1 Aug

Cyanobacteria arrived, cultured in 15x4 ml medium

GFP-dcas9 plasmid ligation (all failed)

2 Aug

Colony PCR for l2, 3, 4

PCR Fragment 1, 2, 3

Plasmid purification

6 Aug

-colony PCR

-standard q5 PCR preperation for gibson

-gibson

- gfp+ dcas9 plasmid again as l3

7 Aug

-gel purification after 75v 45min 0.7 gel

- gibson assembly

- run gel for overnight sgrna PCR product

- restriction endonuclease cut(PstI, HindIII)(2hrs incubation)+ligation for sgrna and petblue

-transformed 3in1, petblue sgrna and L3 into NEB5a

8 Aug

Colony PCR for l3

Run gel for 3in1 (1 smear),

Pick clone

9 August

1. Checked plates for sgrna pet blue mcyi

Sgrna pet blue mcyI2

Sgrna pet blue mcyb 2

3 in 1

Gib 2.1

Gib 2.2

-all no colony.

1. Conducted PCR purification of overnight (14h) restriction endonuclease cut of dcas9 gfp (EcoRI, NsiI)

-50 rxn mixture

1. Conducted ligation

1:3 & 1:5

Vector 250ng

Both 20ul rxn,

-rm temp for 10 min

heat inactive at 65c for 10 min

on ice 90min

-then add ligase again

ligate over weekend.

1. Amplified Fragment1 + 2 + 3

-sgrna I, sgrna B, gfp

For gibson

Fragment 1 2 3 gfp, 65c

Sgrnas 55c

All 50 ul rxn

1. Weight powder, for soc (800ml no glucose/ mgxx) (no yellow)

-LB agar (have yellow) 1800ul

Glucose 9g

1. Run gel for all 6 Fragments (all in)

-75v 45 mins

Ladder use 1kb & 50bp

Loading dye 6x

1. Gel purification -elude using 30ul elution b.

12 August

1. Colony PCR for those without bands reaction

-picked colonies from master plate

1. Performed gibson(gib3) & nebuilder- Scaled down to 10ml rxn- Concentration acc to following nanodrop

-incubation 1h

1. PCR for xgib 1 (Fragment1) gib1 (Fragment1)

xgib 2 (Fragment2) gib2 (Fragment2)

xgib 3 (Fragment3) gib3 (Fragment3)

1. Colony PCR no.24 correct band

(dcas9 gfp) L3

band size 700-800 bp

inoculated 24 Hours (picked from master plate)

1. Transformed gibson 1 gibson 2 gibson 3.2 nebuilder (1)

+ve control petblue nebuilder (2)

dcas9 gfp 1:3

dcas9 gfp 1:5

gibson 3.1

13 August

1. Run gel for previous PCR

1. Checked plates from 12/8

Colony: petblue +ve

dCas9 gfp 1:3

dCas9 gfp 1:5

1. Plasmid purification l3

dCas9-GFP 24

Re cut for Fragment1-3 (gibson assembly)

McyI, Mcyb

14 August

1. Run gel for xgib Fragment 1

-no bands at all

Possibly due to pipetting errors.

1. Started 10ul reaction using gibson concentration according to following gel. (pic gib4)

Reaction: Fragment3: 50ng

Fragment2: 38.73ng

Fragment1: 29.26ng

nebuilder: 5ul

h2o

1. Set up PCR reaction for xgib Fragment2

Xgib Fragment3

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| 98c | 98c | 66c | 72c | 72c | 4c |
| 30sec | 5s | 15s | 1.5min | 2min |  |

1. Transformed gib 4.1 gib 4.2

Overnight ligation sgrna McyI petblue

Sgrna Mcyb PETblue

gib -0.9ul dna

sgrna -2ul

1. Checked plates for previous transformation: gib 3 in 1 -

gib 2 - no colonies

gib 3 - from last year -PETblue stock -yes colonies

1. Picked 6 colonies from petblue

petblue 1-3 -3ml(with amp)

petblue 4-5 -2ml

Master plate -no xgal iptg

1. Run gel for PCR products

Fragment 2, Fragment 3 x gib

Gel purification

Dna concentration on 13/8

15 August

1. Restriction endonuclease cut for Fragment1 Fragment 2 and Fragment 3

Xgib ~6 hr (MluI, BssaI and NheI)

nanodrop before restriction endonuclease cut

1. Set up gibson reaction- Fragment 3 50ng 16ul-- Fragment 2 38.73ng 2.1 ul-- Fragment 1 29.26ng 0.4ul-20ul/xh- Nebuilder 10ul-- H2o 5.9ul-

50c for 2hr (optimized protocol)

1. Plasmid purification of petblue

elute using 30ul

nanodrop

1. Gib 4.1 -petblue mcyb plasmid have 1 colony each

-picked & inoculated

1. PCR purified restriction endonuclease cuts from (Fragment 1, 2, 3 13/8?)

-ligated h3 & 3 in 1

1+3 1:1

1+2+31:1:1

1. Sequencing

16 August

1. Plasmid purification of

Gib4.1 & pet blue mcyb plasmid from 1518

Gib4.1 does not seem to grow

During inoculation

Therefore, low yield.

1. Run gel for Fragment 1+3 & 3in1 (ligated)

1. Intended to amp 1+3 using 3f + 1r and 3r + 1f

1. Intended to check gib4 using 1+2, 2+3, 1+3 primers.

(3) and (4) failed possibily due to PCR errors

-use 3 mins extension 65c other times and maximum

1. Intended to check sgrna mcyb petblue using colony PCR

(failed due to 0.7% gel, not 1.5% gel pic in chelsy’s phone

19 August

1. Colony PCR for mcyb sgrna petblue

(only 1 white colony)

Results positive: band size ~300bp

1. Inoculated 2 3ml tubes of gib4

-gib4.1 & 4.2

From master plate

1. Set up overnight PCR to amp mcyi, Fragment1, Fragment3 xgib,

1. Restriction endonuclease (PstI, HindIII, MluI, BssaI, NheI) cut mcyi (247.8ng), petblue (1000ng), Fragment1 (741ng)- Fragment3 (140ng) overnight [mcyi only added 3ul]

1. Checked neb 3 in 1 using different primers

Checked Fragment 1+3

20 August

1. Run gel for mcyi, Fragment1-3 xgib

-gel purification takara

1. PCR clean mcyi, petblue 2 from overnight restriction endonuclease cut

1. Ligated mcyi petblue, Fragment1+3

-vector 100ng-vector 100ng

1:3 1:1

1. ?

1. Plasmid purification gib6.4

-yield extremely low

1. Picked colonies: 2 nebuilder 3in1.-

2 gib4 -master plate

2 sgrna mcyb petblue-

21 August

1. Plasmid pur for 6 tubes -nebuilder x2, gib4 x2, sgrna mcyb petblue

1. Collected overnight ligation

-amplified Fragment 1+3 wing

Primer Fragment1 f & primer Fragment3 r (vice versa also)

1. Transformed sgrna mcyi petblue and dcas9 gfp (l3)

1. Received sanger sequence results

-99.4% identical

mutation- x

1. Picked 25 colonies from previous l3 plate

-colony PCR 25-49 -one taq

50- sapphire

1. Single cut & double cut 3 in 1 plasmid

-all undesired results

22 Aug

gibson assembly ,incubate 2h

run gel for colony PCR and Fragment 1+3 and the overnight cut of 3 in 1

For colonies with correct band after colony PCR, pick colony from master plate, inoculate

Fragment 1+3, Fragment 2 re cut,

Send mcyb to sequencing

Pick colony from plates if have

Transform gibson products

Ligate Fragment 2 to 1+3

23 Aug

Run gel for PCR dcas9 gfp,

Run gel for PCR 1+3 sgrna mcyi pet blue

26 Aug

Run gel for previous 40 colony PCR

Inoculate l3 with correct band size

Preparation for electroporation

Transform ligation product and 3 in 1 for characterization

27 Aug

Pick clone for previous transformation

3 in 1 plasmid pur using traditional method

Restriction endonuclease cut gib 5 (MluI)

28 Aug

PCR screening mcyi, b

29 Aug

Plasmid purification. Using tradition n takara

Inoculate gib 5, prepare for measurement

30 Aug

I think no lab

3/9

Restriction endonuclease cut dcas9 gfp (EcoRI, PstI)

4/9

Run gel for restriction endonuclease cut dcas9 gfp

5/9

Transformation inoculate pet blue, puc19, 3 in 1, psb1c3 transformants

Plasmid purification 3 in 1 using takara plasmid purification kit

6/9

Plasmid purification 3 in 1 using takara plasmid purification kit

9/9

2 restriction endonuclease cuts (dCas9-GFP-sgRNA)

10/9

Run gel for the colony PCR products (1.5 gel, all in) 65v 45 min 700 or no band

Pick colony and colony PCR

Pour agar plate

take the 2 restriction endonuclease cuts from 37c n run gel (1.5 gel 65v 45 min) shd be ~5500bp and ~3500bp

11/9

PCR clean for 3 in 1 cut

Run gel for the dcas9 gfp sgrna no. 3 restriction endonuclease cut for dcas9 gfp sgrna (XbaI, XhoI), cut the 5500 band (65v 45 min) (big well) (all in)

Gel purification for the above cut gel (gm buffer 650ul)

Run gel for colony PCR yesterday (1.5 gel)

Set ligation mixture (restriction endonuclease cuts of 3 in 1 and mcy dcas9 gfp) 1:1

12/9

Heat inactivate ligation

2. Transform ligation (total dna 60ng)

3. Dilute overnight inoculation, 1 to 3

4. Set restriction endonuclease cut of 3 in 1 and sgrna dcas9 gfp

13/9

Measure od (growth curve) for modelling

16/9

Restriction endonuclease cuts for final product (XhoI, XbaI)

Pick colony for growth curve

17/9

Growth curve

Run gel for the restriction endonuclease cuts, PCR clean, start ligation

Transform ligation mixture

18/9

Transofrmation of final ligation products

2. Od measurements

19/9

pick colony (fat clone)

Transformation final product

20/9

Pick clone

PCR check band

* Correct

Plasmid purification by PETER failed cuz me flip his gel…

23/9

Pick colony of final product

24/9

Plasmid purification

25/9

Nanodrop final product

26/9

Restriction endonuclease cut for final product (PstI)

27/9

Run gel for the restriction endonuclease cuts and final product 24 and 15.

All in for restriction endonuclease cuts,

5ul for final product 24 and 15

65v 50min

Nanodrop final product

30/9

Transformation

final product plasmid 15 17 24 29(bl21)

3/10

Observe green fluorescence by uv exposure

Inoculate

Observe green fluorescence again by uv exposure

* All negative

4/10

Plasmid purification of final product

7/10

Transformation of final products into DH5a

8/10

Prepared 0.1mm nacl, 0.1mm hepes, 0.1mm vitb12, all not sterilized. Picked 10 colonies (final product) , inoculated in 20ml LB

9/10

Filtered Vitamin B12

Autoclave bold3n

Add to agar

10/10

Plasmid purification with ethanol

11/10

Measure Optical density (growth curve)

Transformation of microcystis

Electroporation

Ligation by Peter

Dna purification

14/10

Transformation of previous ligation

15/10

Pick clones, inoculate i12

Standard solution

16/10

Pour plate with kanamycin resistance and chloramphenicol

Takara plasmid purification of insert 1 and insert 2

17/10

Peter ethanol precipitation of final product

Electroporation (natural tranformation protocol)

Ligation mixture (cut with SpelI, xbaI)

Run gel for plasmid purifiction product of i12

Nanodrop i12

Anson adjust pH for bg11

Error: labelling error for plasmid pu

18/10

Pick insert 1 and 2 ,BL21 and NEB5a clones


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:HK SSC/Experiments

![]()

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3AHK_SSC)
[HOME](https://2019.igem.org/Team%3AHK_SSC)

TEAM

[Team Members](https://2019.igem.org/Team%3AHK_SSC/Team)
[Collaborations](https://2019.igem.org/Team%3AHK_SSC/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3AHK_SSC/Description)
[Design](https://2019.igem.org/Team%3AHK_SSC/Design)
[Experiments](https://2019.igem.org/Team%3AHK_SSC/Experiments)
[Model](https://2019.igem.org/Team%3AHK_SSC/Model)
[Notebook](https://2019.igem.org/Team%3AHK_SSC/Notebook)
[Contribution](https://2019.igem.org/Team%3AHK_SSC/Contribution)
[Results](https://2019.igem.org/Team%3AHK_SSC/Results)
[Demonstrate](https://2019.igem.org/Team%3AHK_SSC/Demonstrate)
[Improve](https://2019.igem.org/Team%3AHK_SSC/Improve)
[Attributions](https://2019.igem.org/Team%3AHK_SSC/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AHK_SSC/Parts)
[Basic Parts](https://2019.igem.org/Team%3AHK_SSC/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AHK_SSC/Composite_Part)

[SAFETY](https://2019.igem.org/Team%3AHK_SSC/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AHK_SSC/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AHK_SSC/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AHK_SSC/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AHK_SSC/Hardware)
[Measurement](https://2019.igem.org/Team%3AHK_SSC/Measurement)
[Model](https://2019.igem.org/Team%3AHK_SSC/Model)
[Plant](https://2019.igem.org/Team%3AHK_SSC/Plant)
[Software](https://2019.igem.org/Team%3AHK_SSC/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=HK_SSC)

### Experiment

#### Gene synthesis

Our gblock include a sgRNA specific to mcyB and a GFP with a self-cleaving linker. As part BBa\_K1894001 exceeded the 3000bp requirement of synthesis, ordering was separated into 3 fragments of 1725, 2280 and 2926 bp fragments. Primers for the 3 fragments with 20-25 bp overlaps were also ordered.

![](https://static.igem.org/mediawiki/2019/3/39/T--HK_SSC--frag1.png)
![](https://static.igem.org/mediawiki/2019/7/7f/T--HK_SSC--frag2.png)
![](https://static.igem.org/mediawiki/2019/a/ad/T--HK_SSC--frag3.png)

Fig. 1 Design of fragment 1,2 and 3

![](https://static.igem.org/mediawiki/2019/8/83/T--HK_SSC--SGRNA.png)

Fig. 2 Design of sgRNA

![](https://static.igem.org/mediawiki/2019/6/60/T--HK_SSC--GFPonly.png)

Fig. 3 Design of GFP

![](https://static.igem.org/mediawiki/2019/0/01/T--HK_SSC--Primer.png)

Fig. 4 Design of Fragment 1 primer

#### Protocols

#### gBlock Resuspension

1.Resuspend pellets in nuclease-free water according to manufacturer protocol
2.Store at 4°C.

#### Constructing pSB1C3-GFP-dCas9 plasmid

1. Extract part BBa\_K1689013 from distribution kit according to iGEM protocols
2. Cut BBa\_K1689013 with EcoRI and NsiI according to manufacturer protocol
3. Incubate at 37°C for 5-15 mins or overnight. Ligate GFP-linker complex to cut BBa\_K1689013 according to manufacturer protocol
4. Incubate at room temperature for 10 minutes or overnight. Heat inactivate ligase according to manufacturer protocol and transform to DH5α according to iGEM protocol
5.  Spread onto chloramphenicol plates. Pick clones and perform colony PCR according to manufacturer protocol
6. Perform plasmid purification according to manufacturer protocol

#### Constructing pETBlue2-sgRNA plasmid

1. Cut pETBlue-2 with PstI and HindIII according to manufacturer protocol3. Incubate for 5-15 mins or overnight.
2. Ligate sgRNA to pETBlue-2 according to manufacturer protocol4. Incubate at room temperature for 10 minutes or overnight.
3. Heat inactivate ligase according to manufacturer protocol and transform to DH5α according to iGEM protocol5. Spread onto ampicillin plates with X-gal and IPTG.
4. Perform blue-white screening and inoculate recombinant clones in 3 mL of LB with 3uL of ampicillin.
5. Pick clones and perform colony PCR according to manufacturer protocol 6.
6. Perform plasmid purification according to manufacturer protocol 7.

#### Constructing pETBlue2-sgRNA-dCas9-GFP plasmid

1. Cut pSB1C3-GFP-dCas9 with EcoRI and PstI according to manufacturer protocol 3. Incubate at 37°C for 5-15 mins or overnight.
2. Cut pETBlue2-sgRNA with EcoRI and PstI according to manufacturer protocol. Incubate for 5-15 mins or overnight.
3. Ligate pSB1C3-GFP-dCas9 to cut pETBlue2-sgRNA according to manufacturer protocol 4. Incubate at room temperature overnight.
4. Heat inactivate ligase according to manufacturer protocol and transform to DH5α according to iGEM protocol 5. Spread onto ampicillin plates with X-gal and IPTG.
5. Perform blue-white screening and inoculate recombinant clones.
6. Pick clones and perform colony PCR according to manufacturer protocol 6.
7. Perform plasmid purification according to manufacturer protocol 7.

#### Constructing BBa\_K1894001 shuttle vector

1. Add overlaps to fragments 1, 2 and 3 using PCR according to manufacturer protocol 8.
2. Assemble fragments 1, 2 and 3 using Gibson assembly according to manufacturer protocol. Incubate for 1.5 hours.
3. LPerform PCR purification according to manufacturer protocol 10.

#### Constructing Final Product

1. Cut pETBlue2-sgRNA-dCas9-GFP using XbaI and XhoI according to manufacturer protocol3. Incubate overnight.
2. Cut BBa\_K1894001 using XbaI and XhoI according to manufacturer protocol3. Incubate overnight.
3. Ligate pETBlue2-sgRNA-dCas9-GFP to BBa\_K1894001 at a 1:1 ratio according to manufacturer protocol4. Incubate at room temperature overnight.

#### Culturing Microcystis aeruginosa UTEX-2388

1. Pipette 5 mL of bacteria into 200 mL of Bold 3N medium.
2. Culture at room temperature under bright light with 12-hour intervals.

#### Xia Men natural trasformation

1. Culture cells until Microcystis Aeruginosa UTEX2388 until OD730= 0.25-0.35.
2. Collect cells by centrifugation.
3. Wash cells using 10mL 10 mmol/L NaCl
4. Concentrate cells by centrifugation
5. Add fresh Bold 3N medium in 1/10 the original volume
6. Add 2-3 ug plasmid and mix.
7. Incubate overnight in the dark for selection.
8. Spread the mixture onto solid bold 3N medium covered with Millipore filters, culture it for 20h under normal growth conditions.
9. Transfer Millipore filters to solid Bold 3N kanamycin 15ug/mL of culture for 7-10 days.
For culturing transformants:
10. Pick colonies into 2mL Bold 3N liquid medium (containing 15ug/mL kan), often shaking culture for 7 days.

#### Electroporation

1. Centrifuge and concentrate cells at a density of 10-9 cells per mL
2. Wash cell pellet for 3 times in 500μl of 0.1 mM cold HEPES (pH 7.2)
3. Chill on ice
4. Add 2 to 3 ug DNA to cells on ice to allow for pre-pulse contact at low temperature for 1- 2h
5. 40μl cell construct suspension in a pre-chilled sterile cuvette (with 2-mm gap between the electrodes)
6. (The cells were electroporated using BioRad Gene Pulser equipped with a 25-µF capacitator and resistance was fixed at 400 ohms.)
7. Add 2mL of sterile cold Bold 3N medium immediately after first pulse. Mix well.
8. Culture in 10mL bold 3N media without kan with continuous white light for 1 day.
9. Centrifuge culture.
10. Spread the pellet onto solid Bold 3N medium with kanamycin (15ug/mL).
11. Grow plates at room temperature with continuous white light.
12. Check plates under dissecting microscope.

#### Microcystin test kit

1. Collect a 1 mL sample in a 2 ml centrifuge tube. If the sample is turbid, centrifuge or filter before testing.
2. Before use, remove the test strip and return the microwell to room temperature.
3. Add 80 uL of sample into microwell and pipette up and down 3-5 times. Incubate for 5 min, then insert test strip into microwell.
4. After 5-8 minutes, observe the test strip. The results will become invalid after 8 minutes.

#### Reference

“Resuspending GBlocks Gene Fragments.” Tips for Working with GBlocks Gene Fragments, Integrated DNA Technologies, 25 Apr. 2017, https://sg.idtdna.com/pages/education/decoded/article/tips-for-working-with-gblocks-gene-fragments.

“DNA Kit Plate Instructions.” Help:2019 DNA Distribution, Standard Registry of Biological Parts, http://parts.igem.org/Help:2019\_DNA\_Distribution.

“NEBcloner.” NEBcloner, New England Biolabs, https://nebcloner.neb.com/#!/redigest.

“Ligation Protocol with T4 DNA Ligase (M0202).” New England Biolabs: Reagents for the Life Sciences Industry, New England Biolabs, https://international.neb.com/Protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202.

“Single Tube Transformation Protocol.” Help:Protocols/Transformation, Standard Registry of Biological Parts, https://parts.igem.org/Help:Protocols/Transformation.

“Protocol for OneTaq® 2X Master Mix with Standard Buffer (M0482).” New England Biolabs: Reagents for the Life Sciences Industry, New England Biolabs, 6 Sept. 2012, https://international.neb.com/protocols/2012/09/06/protocol-for-onetaq-2x-master-mix-with-standard-buffer-m0482.

“Protocol.” Takara MiniBest Plasmid Purification Kit Ver 4.0 Product Manual, TaKaRa Bio Inc., https://takara.co.kr/file/manual/pdf/9760e.v1706Da.pdf.

“Protocol for Q5® High-Fidelity 2X Master Mix.” New England Biolabs: Reagents for the Life Sciences Industry, New England Biolabs, 7 Dec. 2012, https://international.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492.

“Gibson Assembly® Protocol (E5510).” New England Biolabs: Reagents for the Life Sciences Industry, New England Biolabs, 11 Dec. 2012, https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510.

“Protocol.” TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 Product Manual, TaKaRa Bio Inc., https://takara.co.kr/file/manual/pdf/9761\_e.v1306Da.pdf.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:HK SSC/Results

![]()

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3AHK_SSC)
[HOME](https://2019.igem.org/Team%3AHK_SSC)

TEAM

[Team Members](https://2019.igem.org/Team%3AHK_SSC/Team)
[Collaborations](https://2019.igem.org/Team%3AHK_SSC/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3AHK_SSC/Description)
[Design](https://2019.igem.org/Team%3AHK_SSC/Design)
[Experiments](https://2019.igem.org/Team%3AHK_SSC/Experiments)
[Model](https://2019.igem.org/Team%3AHK_SSC/Model)
[Notebook](https://2019.igem.org/Team%3AHK_SSC/Notebook)
[Contribution](https://2019.igem.org/Team%3AHK_SSC/Contribution)
[Results](https://2019.igem.org/Team%3AHK_SSC/Results)
[Demonstrate](https://2019.igem.org/Team%3AHK_SSC/Demonstrate)
[Improve](https://2019.igem.org/Team%3AHK_SSC/Improve)
[Attributions](https://2019.igem.org/Team%3AHK_SSC/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AHK_SSC/Parts)
[Basic Parts](https://2019.igem.org/Team%3AHK_SSC/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AHK_SSC/Composite_Part)

[SAFETY](https://2019.igem.org/Team%3AHK_SSC/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AHK_SSC/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AHK_SSC/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AHK_SSC/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AHK_SSC/Hardware)
[Measurement](https://2019.igem.org/Team%3AHK_SSC/Measurement)
[Model](https://2019.igem.org/Team%3AHK_SSC/Model)
[Plant](https://2019.igem.org/Team%3AHK_SSC/Plant)
[Software](https://2019.igem.org/Team%3AHK_SSC/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=HK_SSC)

Results

We have successfully constructed our 12kbp plasmid and expressed the dCas9 protein. Here are the results:

# 1. Plasmid construction

After various restriction enzyme cuts and ligation, we have successfully constructed our 12kbp plasmid.

![](https://static.igem.org/mediawiki/2019/8/86/T--HK_SSC--results_gelpic.png)

**Fig1. The first and last lanes are NEB 2-log ladder. Lanes 2 and 3 are linearized plasmids of our final plasmid, showing the band size about 12kbp.**

# 2. Successful Transformation

We have successfully transformed our plasmids into Microcystis and spread onto selective plates, showing that the plasmid we constructed is compatible in both E.coli and Microcystis. Besides, our transformed cells demonstrated kanamycin resistance. These are living colonies as we checked the plates under microscope every day. We ensured that the cells were not only dead cell bodies buy transfering them to new selective plates once in two weeks.

![](https://static.igem.org/mediawiki/2019/6/62/T--HK_SSC--Microcystis_plate.jpg)

**Fig2. Microcystis Aeruginosa UTEX2388 transformed with our 12kbp plasmid spread onto a Kanamycin plate.**

![](https://static.igem.org/mediawiki/2019/b/b3/T--HK_SSC--Final_product.jpeg)

**Fig3. E.coli DH5α transformed with our 12kbp plasmid and spread onto a Kanamycin plate.**

# 3. Plasmid expression

We used Microcystin-LR detection kit (WRZ001) from Guangzhou Oasisbio chemistry Technology Co., Ltd. We believed that our dCas9-sgRNA complex has been successfully been expressed. This is because the Microcystin-LR concentration in our transformed Microcystis was lower than 0.002mg/L, while our control set ups have a higher concentration.

![](https://static.igem.org/mediawiki/parts/8/87/T--HK_SSC--Test_paper.jpg)

**Fig 4. Microcystin detection kit sample**

![](https://static.igem.org/mediawiki/parts/7/70/T--HK_SSC--results.jpeg)

**Fig5. 1st test (from left): Culture of Microcystis 3 weeks after transformation
2nd test: Water
3rd test: Culture of unsuccessful Microcystis transformation after 3 weeks
4th test: Positive control of Microcystis culture that has not been transformed**

The successful transformation of Microcystis culture showed Microcystin concentration less than 0.002mg/L. This shows a decrease in Microcystin toxin.

# 4. GFP Expression

Through UV excition, we the green flourescence protein has been successfully expressed, demonstrating flourescence properties.

![](https://static.igem.org/mediawiki/2019/7/74/T--HK_SSC--greenlmao.jpg)

**Fig 6. Cell cultures with disired plasmid under UV excitation.**

# 5. Quantitative Real-Time PCR

We will be conducting quantitative real-time PCR in the future for further verification of our results.