IGEM:Hong Kong HKUST/Investigations/Effect of ATP concentrations on ligation efficiency

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Contents

Authors

Abstract

ATP-dependent T4 Ligase is a powerful tool in synthetic biology. This experiment was conducted to find the correlation between ATP supplement and ligation velocity. Quantitative results are obtained by counting the colony-forming unit (CFU) formed from the transformation of ligated DNAs. From three different ATP concentration used in the ligation, 1mM, 2mM, and 4mM, it is observed that the higher the ATP concentration, the less CFU are formed. This experiment, backed by literature, showed that high concentration of ATP interferes with ligation process. This information would be useful in determining the optimum concentration of ATP for T4 Ligase activity.

Introduction

This study is to investigate the relationship between ATP and T4 ligase activity. It is important as it helps determine the optimum environment for ligation which is essential in basically every genetically engineering process. The approach employed to carry this investigation was using bacteria that were already transformed with RFP-pSB1C3, and extracting the plasmid from them. The plasmids were cut by restriction enzymes(EcoRIHF & PstIHF), then by putting the insert and backbones in different concentration of ATP. Afterward, putting the new plasmids into competence cells, and compare the number of colonies forming units between different concentration of ATP, which represents the amount of ligated plasmid produced.

E.coli was used as our host of plasmid because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.pSB1C3 was used as the backbone of plasmid since it is a high copy number plasmid carrying.

The experiment result was expected to be the more ATP there is ,the faster the ligation process. The concentration was decided to be 1mM, 2mM(by doubling 1 mM), and 4mM(by doubling 2 mM). The reason why 1mM was chosen is that 1X of T4 ligase buffer from New England Lab contains 1mM of ATP buffer.

Procedure

1. Preparation of Chloramphenicol(CHL) LB plates: 500µl of 150mg/ml of Chloramphenicol was added to 500 ml of LB broth solution. All plates were then stored in refrigerator.

2. Inoculation of bacteria in broth: E.coli SURE with RFP-pSB1C3 was obtained from four different colonies and inoculated to the 15 ml Falcon tube with 5 ml CAM LB broth. The tubes were incubated under 37ºC for 16 hours, 200 rpm. The inoculated bacteria were named RFP-pSB1C3 1-4.




3. Take out the inoculated RFP-pSB1C3 (1,2,3,4) at 9:00am
4. Centrifuge the RFP PSB1C3 (1,2,3,4) for 5 minutes, using 3500 rpm speed.

5. MiniPrep :
Plasmids were extracted using Gene Tech GTpure Plasmid MiniPrep Purification Kit. DNA then eluted in 50 μL DE Buffer (2 times)


6. NanoDrop : Use DE Buffer as Blank

7. Digestion : EcoRI-HF & PstI-HF
Use Cutsmart buffer
Digestion Composition :
- 1 ug DNA (1000 ng)
- 2 uL of Cutsmart buffer
- 0.5 uL of EcoRI-HF
- 0.5 uL of PstI-HF
- ddH2O = Adjust volume to 20 uL
Incubate for 1 hour, 37 degree Celcius.

8. Gel Electrophoresis :
0.8% Agarose Gel, 20 ml, 6 wells

WELL 1 (LADDER):
- 1 uL Loading Dye
- 1 uL GenRuler 1 kb Ladder
- 8 uL ddH2O

WELL 2 (NEGATIVE CONTROL): - 1 uL of Uncut Plasmid RFP-pSB1C3(2) (124.8 ng/lane)
- 1 uL Loading Dye
- 8 uL ddH20

WELL 3-6 (DIGESTION PRODUCT)(1ug/lane):
- 20 uL of DNA
- 2.2 uL of 10X Loading Dye


9. Gel Extraction & Purification
Insert and backbone were extracted out from the gel using Favorgen FavorPrep GEL/PCR Purification Kit. The DNA is then eluted in 50 μL of Elution Buffer

10. NanoDrop



11. Making the ATP Stock

Make 10 mM ATP stock for 1 ml by doing serial dilution.
1. Weigh out 0.06 grams of powdered ATP and dilute it using 1 ml of water.
2. Pipette 100 µl of the diluted ATP powder and transfer it into a micro centrifuge tube.
3. Add 900 µl of double distilled water to make the 10mM ATP stock.

The ATP solution then sterilized with filter sterilization

12. Ligation

Make the recipe for ligation. Each person in the team will use 4 micro centrifuge tube to create the control, 1mM ATP, 2mM ATP, and 4 mM ATP. The amount of insert and backbone added varies due to the different amount of concentration of nucleic acid in each set.

The ratio of insert to backbone must be 3:1. And since the length of the insert and backbone is 1 kb to 2 kb respectively, the amount of insert must be 30 ng and backbone must be 20 ng. ( total amount of both insert and backbone is ~50 ng)


Control
1mM ATP
2mM ATP
4mM ATP
Insert
30.0 ng
30.0 ng
30.0 ng
30.0 ng
Backbone
20.0 ng
20.0 ng
20.0 ng
20.0 ng
10x T4 buffer (contain 1mM of ATP) (µl)
-
5.0
5.0
5.0
10 mM ATP (µl)
-
-
5.0
15.0
T4 Ligase (µl)
0.5
0.5
0.5
0.5
ddH2O
Adjust to total volume
Adjust to total volume
Adjust to total volume
Adjust to total volume
Total volume (µl)
50.0
50.0
50.0
50.0

Incubate in room temperature for 50 minutes

13. Transformation
Transform the plasmid into the competent cell DH5α (high efficiency) with 1hr recovery period.

14. Culture
Grow transformed cells on the Chloramphenicol LB plates for 16 hours in 37°C using spread plate method.

Results

  • Step Number 6 NanoDrop Result :
Concentration
A260/A280
A260/A230
RFP pSB1C3 (1)
228.1
1.93
2.12
RFP pSB1C3 (2)
124.8
1.91
1.91
RFP pSB1C3 (3)
220.8
1.90
1.65
RFP pSB1C3 (4)
219.2
1.94
2.26
  • Step 10 NanoDrop result :



Concentration (nanogram/microliter)
A260/A280
A260/A230
RFP Insert 1
5.0
3.37
0.17
RFP Insert 2
4.0
2.74
0.07
RFP Insert 3
3.4
4.19
0.12
RFP Insert 4
7.0
2.28
0.04
pSB1C3 Backbone 1
7.1
2.51
0.21
pSB1C3 Backbone 2
5.9
3.09
0.13
pSB1C3 Backbone 3
3.3
10.81
0.40
pSB1C3 Backbone 4
5.8
3.63
0.06


  • Step 13 Transformation result :


Figure 1. RFP-pSB1C3 (1), Clockwise from top left: Control, 1mM ATP, 4mM ATP, 2mM ATP




Figure 2. RFP-pSB1C3 (2), Clockwise from top left: Control, 1mM ATP, 4mM ATP, 2mM ATP




Figure 3. RFP-pSB1C3 (3), Clockwise from top left: Control, 1mM ATP, 4mM ATP, 2mM ATP



Figure 4. ATP Concentration Effect to Ligation Rate



The result of our experiment is that higher ATP concentration reduce ligation rate. We can see the ligation rate from the colony of bacteria that grow within the LB medium. As the ATP concentration increase from 1mM, 2mM, to 4mM, the number of bacteria colony decrease, hence the ligation rate decrease.


Discussion

In the contrary to the hypothesis, results of experiment shown that higher ATP concentration reduce ligation rate. A possible explanation is due to the high concentration of ATP, adenylated DNA intermediate was accumulated exceeding threshold amount. In this condition, ligase could not perform its usual function to form phosphodiester bond between DNAs because most of the ligase are on the adenylated form. Lack of phosphodiester bond cause the inhibition of ligation and in the end reduce the ligation rate.

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