Team:HK SSC/Experiments

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.

Fig. 1 Design of fragment 1,2 and 3

Fig. 2 Design of sgRNA

Fig. 3 Design of GFP

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.