Team:Korea U Seoul/Project/sub proc result

Our experiments consists of 2 parts, the pili display and the GFP pili display. First we expressed pili on Corynebacterium glutamicum to see whether this is possible, and then we tried to insert the GFP into the subunit to confirm the possibility of protein display. We also tried to measure the amount of pili that has been produced.
Gibson assembly was used to assemble the plasmids.

1. pili expression in Corynebacterium glutamicum

pEKEx2-pilin A gene cluster, E1

pEKEx2-pilin A gene cluster, E1 is a plasmid that has pilin A gene cluster insert and pEKEx2 shuttle vector.

* Method of pili A expression

1. Design PCR primers
2. PCR of pilin A gene cluster with 5’ & 3’ overhang that matches pEKEx2 vetcor’s corner from genomic DNA of Corynebacterium diphtheriae
3. PCR of pEKEx2 with 5’ & 3’ overhang that matches pilin A gene cluster insert’s corner
4. Gibson assembly of two PCR products, pilin A gene cluster PCR products & pEKEx2 PCR products

- Then we can make a gibson assembly products, pEKEx2-pilin A gene cluster, E1

5. Transformation gibson assembly products into E.coli DH5a for amplification
6. Plasmid prep & Transformation E1 into C. glutamicum ATCC 13032

- Then we can make C.glutamicum that has pili A

Result of E1

(left : C. glutamicum-pEKEx2, Right : C. glutamicum-E1)

We made E1 and expressed it in the cell, which resulted in aggregation of the cells. The aggregation seems to have occured by the pili production.

2. GFP pili expression

To ascertain the formation of the GFP pili we designed 2 controls, each expressing GFP pili and GFP only. The following is the structure of the spa A gene.

(spa A gene structure : Hung Ton-That, Luciano A. Marraffini and Olaf Schneewind, Sortases and pilin elements involved in pilus assembly of Corynebacterium diptheriae, Molecular Microbiology (2004) 53(1), 251-261)

We tried to make the GFP pili by inserting GFP into the known handling site of the spa A gene.

pEKEx2-GFP pili, E2

pEKEx-GFP pili, E2 is a plasmid that has modified pilin A gene cluster by inserting GFP into spa A and pEKEx2 shuttle vector.

* Original method of GFP pili expresssion

1. Design PCR primers
2. PCR of pEKEx2-pilin A gene cluster with 5’ & 3’ overhang that matches corner of GFP and with exclusion of spa A middle part that is converted by GFP
3. PCR of GFP with 5’ & 3” overhang that matches corner of spa A middle part that is converted by GFP
4. Gibson assembly of two PCR products

- Then we can make a gibson assembly products, pEKEx2-GFP pili, E2

5. Transformation gibson assembly products into E.coli DH5a for amplification
6. Plasmid prep & Transformation E2 into C. glutamicum ATCC 13032

- Then we can make C.glutamicum that has GFP pili

* Alternative method of GFP pili expression

1. Design PCR primers
2. PCR of promoter & spa A of pilin A gene cluster with 5’ & 3’ overhang that matches corner of pEKEx2 & GFP and with exclusion of spa A middle part that is converted by GFP
3. PCR of spa A and srt A, spa B, spa C of pilin A gene cluster with 5’ & 3’ overhang that matches corner of pEKEx2 & GFP and with exclusion of spa A middle part that is converted by GFP
4. PCR of pEKEx2 with 5’ & 3’ overhang that matches corner of promoter and spa C of pilin A gene cluster
5. PCR of GFP with 5’ & 3” overhang that matches corner of spa A middle part that is converted by GFP
6. Gibson assembly of four PCR products.

- Then we can make a gibson assembly products, pEKEx2-GFP pili, E2

7. Transformation gibson assembly products into E.coli DH5a for amplification
8. Plasmid prep & Transformation E2 into C. glutamicum ATCC 13032 Then we can make C.glutamicum that has GFP pili

pEKEx2-GFP control, C1

1. Design PCR primers
2. PCR of pEKEx2-pilin A gene cluster with 5’ & 3’ overhang that matches corner of GFP and with exclusion of spa A, srt A, spa B and spaC
3. PCR of GFP with 5’ & 3” overhang that matches corner of pEKEx2 and promoter of pilin A gene cluster
4. Gibson assembly of two PCR products.
5. Gibson assembly of four PCR products.

- Then we can make a gibson assembly products, pEKEx2-GFP control, C1

6. Transformation gibson assembly products into E.coli DH5a for amplification
7. Plasmid prep & Transformation C1 into C. glutamicum ATCC 13032 Then we can make C.glutamicum that expresses GFP by using promoter of pilin A gene cluster

Result of E2 and C1

We already designed the experiment but we did not have enough time to go through cloning.

1. E.coli Competent Cell Preparation and Transformation

Preparing the competent cells.

Reagent: TSS (Transformation and Storage Solution for chemical transformation)

• 85 % LB medium
• 10 % PEG (wt/vol, MW 8000)
• 5 % DMSO (vol/vol )
• 50 mM MgCl2 (pH 6.5)
  
  
  

Autoclave or filter sterilize. Store at 4 oC for < 2 weeks.

1. Streak the cell stock on a LB plate (added antibiotic if cells have antibiotic resistant). Incubate the plate at 37oC overnight.
2. Pick a single, well-isolated colony and inoculate it into 5 ml of LB broth (plus antibiotic). Incubate at 37 oC overnight with shaking at 220 rpm.
3. Transfer 1 ml of the saturated overnight culture to a sterile 500-ml flask containing 100 ml of LB medium (do not add antibiotic at this step). Incubate the cells at 37 oC with the shaking at 220 rpm, until OD600 reach 0.5 ; this usually takes 2.0-2.5 hr. Check the OD frequently when it gets beyond 0.2 to avoid overgrowth.
4. When the culture reaches an OD600 of 0.5, chill the flask on the ice for 20 min and then collect the cells by centrifugation at 1500 rpm for 5 min at 4 oC.
5. Resuspend the cells in 10 ml of ice-cold TSS solution. Now the competent cells are ready to be transformed.
6. Aliquot 150 ul competent cells to 1.5ml tube. If they are not immediately used, cells can be stored at 4 oC for maximum of 6 hr without significant loss of competency. The same competent cells can also be stored at -70 oC for long-term storage (pre-treat with liquid nitrogen or dry ice).

- (p.s.) Competent cells should give a minimum of 1x106 transformants per ug of plasmid DNA. Transformation frequency of frozen cells is 30 % of that of the fresh cells, when used within two months.

Transforming the cells.

1. Add DNA (under 20 ul ) to ice cold 150 ul competent cells. Thaw -70 oC competent cells at 0oC. (You can transform smaller portions of cells (eg. 75 ul or 50 ul), but you should decrease the volumes of DNA and media proportionately.)
2. Incubate on ice for 30 min. with occasional mix.
3. Heat shock at 42 oC for 2 min.
4. After heat shock, put on ice for 2 min.
5. Add 0.8 ml LB broth.Many transformation protocols indicate that you should allow cells to recover in SOC (2% Bactotryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose). I think this improves the number of colonies you get.
6. Shake and incubate at 37oC for 60 min. ( you can put 1.5 ml tubes in a big container or breaker to shake)

Reference

Chung, C. T. et al. (1989) One-step preparation of competent E. coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86:2172-2175.

2. E.coli plasmid prep by using miniprep kit

1. Corynebacterium glutamicum Competent cells Preparation

Reagents

• Corynebacterium glutamicum
• LB broth
• Cold 10% glycerol
• Cold TG-Buffer (autoclaved)
• 1mM Tris, 10% glycerol in DW, pH 7.5

Procedure

1. Streak the cell stock on a LB plate. Incubate the plate at 30oC overnight.
2. Pick a single, well-isolated colony and inoculate it into 3 ml of LB broth. Incubate at 30 oC overnight with shaking at 220 rpm.
3. Transfer 1 ml of the saturated overnight culture to a sterile 500-ml flask containing 100 ml of LB medium. Incubate the cells at 30 oC with the shaking at 220 rpm, until OD600 reach 1.75 ; this usually takes more than 6 hr.
4. When the culture reaches an OD600 of 1.75, chill the flask on the ice.

- All procedure must be taken at 0 oC, and centrifuge must be taken at 4 oC after OD600 reaches 1.75

5. Collect the cells in sterilized Falcon tube by centrifugation at 3600 rpm for 10 min at 4 oC.
6. Resuspend the cells in 20ml of cold TG buffer. (In this period, collect all cells in one tube)
7. One more ‘5 to 6’ cycle
8. Centrifugation at 3600 rpm for 10 min at 4 oC.
9. Resuspend the cells in 20ml of cold 10% glycerol
10. One more ‘8 to 9’ cycle and Centrifugation at 3600 rpm for 10 min at 4 oC.
11. Resuspend the cells in 1ml of cold 10% glycerol. Now the competent cells are ready to be transformed.
12. Aliquot 150 ul competent cells to 1.5ml tube. If they are not immediately used, cells can be stored at at -80 oC for long-term storage (pre-treat with liquid nitrogen or dry ice).

2. Electroportaion of Corynebacterium glutamicum Cell

Reagents

• Competent cell (100ul)
• LB Broth and media(antibiotics 15ug/ml)
• Cold 10% glycerol

Procedure

1. Prepare the 46℃ pre-warmed LB 2ml.
2. Thaw -70 oC competent cells at 0oC.
3. Add DNA to ice-cold 100ul competent cell and tranfer it to ice-cold 0.2cm gene pulser-cuvette.
4. Add cold 10% glycerol 200ul. (cold 10% glycerol should not be mixed with cells.)
5. Put dried Cuvette into gene pulser.
6. Pulse the electric current at 2.5kV 25uF 200ohm.
7. After pulse, mix with pre-warmed LB broth 2ml
8. Incubate it at 30℃ 50min with shaking at 200rpm.
9. Streak it at LB plate that has antibiotics. Incubate the plate at 30oC two days.

3. Corynebacterium glutamicum Plasmid preparation

Reagents

• Corynebacterium glutamicum
• LB Broth
• Plasmid mini-prep kit

Procedure
Use plasmid miniprep kit with lysosyme 15mg/ml added resuspension buffer.

Reference

Lothar Eggeling, Michael Bott, Handbook of Corynebacterium glutamicum, 2005, CRC Press Taylor&francis Group. Modified by Jade Minseob Yoo, Korea University, 2014.