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.

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.

• 20mM Tris-HCl buffer, pH 8.0.
• 20mM Tris-HCl buffer, pH 8.0 + 1M Imidazole.
• 1M NaHCO3.
• 1M CaCl2.
  
  
  

Enzyme

Using Protino® Ni-NTA Columns for His-tag protein purification, purify the enzymes.

These enzymes is diluted in 20mM Tris-HCl buffer, pH 8.0 + 10mM Immidazole at 4°C.

Procedure

• Blank Determination
Add 0.5 ml of 1M CaCl2 to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Immediately start a stop watch and record the time and pH up to 20 minutes.
• Enzyme Determination
Add freshly diluted enzyme to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Add Add 0.5 ml of 1M CaCl2. Immediately start a stop watch and record the time and pH up to 20 minutes.

Method

The electrometric method of Wilbur and Anderson (1948) in which the time required (in seconds) for a saturated CO2 solution to lower the pH of 0.012 M Tris⋅HCl buffer from 8.3 to 6.3 at 0°C is determined. The time without enzyme is recorded at T0; with enzyme, T.

A unit of activity = (2×(T_0-T))/T

Reagents

0.02 M Tris⋅HCl buffer, pH 8.0. Store in an ice bath at 0-4°C before and during use.
Carbon dioxide saturated water. Bubble CO2 gas through 200 ml ice cold water for 30 minutes prior to assay. During saturation process, store water at 0-4°C in an ice bath.

Enzyme

Using Protino® Ni-NTA Columns for His-tag protein purification, purify the enzymes.
These enzymes is diluted in 20mM Tris-HCl buffer, pH 8.0 + 10mM Immidazole at 4°C.

Procedure

• Blank Determination
Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. Withdraw in a 5 ml syringe, 4 ml of chilled CO2 saturated water and add to Tris buffer. Immediately start a stop watch and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T0.


• Enzyme Determination
Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. Add 0.1 ml of freshly diluted enzyme. Quickly add 4 ml of CO2 saturated water and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T.