Team:NTU-Singapore/Wetlab/Protocols

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I) MATERIALS:<br>
 
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1) Preparation of AI-2:<br>
 
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Pure AI-2 is very difficult to harvest or synthesize. However, as a quorum sensing messenger, AI-2 is present in intercellular medium at considerable amount. In this project, AI-2 was indirectly harvested by obtaining cell supernatants at different time points of cell growth. Glucose was added to the growth medium as it promotes AI-2 production significantly (Liang Wang, 2005).
 
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A colony of E. coli W3110 was grown overnight in LB for 16 hours at 37 oC. The overnight culture was then diluted in LB plus 0.8% glucose to an optical density at 600 nm (OD600) of 0.02. The cells were incubated at 37oC with shaking at 225 rpm in 500ml Erlenmeyer flask. Cell aliquots were removed at each time point for measurement of the OD600. The measured values were shown in the following table:
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='''Protocols'''=
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{|border="1"
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=='''Creation of New Biobrick parts'''==
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|Time (hrs)
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To create a new biobrick part, the first step is to identify the gene of interest. In this project, our genes of interest are the E7 production gene, Immunity gene, and Lysis gene that is obtainable from the plasmid ColE7-K317 in BW Escherichia Coli strain. Another gene of interest is the LsrA promoter that is obtained from MG1655 Escherichia Coli strain. Information on the genome of interests is extracted from relevant publications and websites such as NCBI net. <br>
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|OD of W3110
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|-
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|0
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|0.002
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|1
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|0.047
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|-
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|2
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|0.112
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|-
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|3
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|0.341
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|-
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|4
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|0.628
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|5
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|0.921
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|-
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|6
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|1.032
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|-
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|7
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|1.172
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|8
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|1.252
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|}
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Based on the data, a graph of OD600 versus time was constructed as below:
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For example the gene of interest – Lysis gene: <br>
 +
1. A pair of DNA primers, 10 ~ 15 nucleotides in length are designed and synthesized to flank the forward 3' – 5' end and reverse 5' – 3' end of the gene of interests. The primers also contain blunt ends of the EcoRI, XbaI, SpeI & PstI restriction sites. These primers will hook on to the double strand DNA by complementary pairing and serve as initiation and termination site of DNA amplification.<br><br>
-
[[Image:ODW3110.JPG]]
+
2. Amplification of the lysis gene occurs by PCR amplification. A gel electrophoresis is run, followed by a gel extraction of the specific interest.<br><br>
-
Maximal AI-2 production activity is typically observed during mid- to late exponential phase (Surette, 1998). Therefore, 4ml of samples corresponding to 2, 3, 3.333, 3.667, 4, 6 time points were collected and centrifuged at 13,200 rpm for 10 min in a microcentrifuge to separate cell pellets from supernatants.  Cleared supernatants were filtered using 0.2 µm pore size filters to remove unwanted big proteins. Supernatants were stored at -20oC.
+
 
 +
3. The lysis gene is then purified using the PCR purification kit. Purified DNA is tested for concentration and purity using NanoDrop. <br><br>
 +
 
 +
4. Next, the purified DNA is digested using EcoRI and PstI restriction enzymes to generate a standard Biobrick part with the gene sequence:<br>
 +
 +
'''EcoRI – XbaI - Lysis gene – SpeI – PstI'''<br><br>
 +
 
 +
[[Image:Cycle_mindmap.jpg|thumb|600px|center|Creation of New BioBricks]]
 +
 
 +
=='''Digestion and Ligation of NTU@iGEM System'''==
 +
To ligate 2 Biobrick parts together, firstly we identify one part to be "vector" and the second part to be "insert". There are 2 possibility of ligation:<br>
 +
*Case I:  Insert in front – Vector behind <br>
 +
*Case II: Vector in front – Insert behind <br>
 +
 
 +
===Case I:  Insert in front – Vector behind===
 +
1.Digest Insert gene with EcoRI & SpeI and Vector gene with SpeI & PstI. <br>
 +
Purify the digested products with PCR Purification Kit and test for concentration and purity using NanoDrop. <br><br>
 +
2.Perform a gel electrophoresis of the purified products, followed by a gel extraction using PCR Gel Extraction Kit. <br><br>
 +
3.Fuse the Insert gene to the Vector gene to form a circular plasmid DNA using T4 DNA Quick Ligase. <br>
 +
Transform the DNA plasmid into Top 10 Competent Cells and select using appropriate antibiotic treatment. <br>
 +
Vector gene contain antibiotic resistant gene that confers resistance to the cells that are successfully transformed.<br><br>
 +
4.Extract the ligated DNA using MiniPrep Kit and digest the resultant DNA using EcoRI & PstI. <br><br>
 +
5.Lastly, perform a gel electrophoresis for confirmation. Check if the nucleotide length from the gel run matches the hypothetical nucleotide length of the digested product. If the result matches, ligation is successful.<br><br>
 +
 
 +
===Case II:  Vector in front – Insert behind ===
 +
1. Digest Insert gene with XbaI & PstI and Vector gene with EcoRI & XbaI. Purify the digested products with PCR Purification Kit and test for concentration and purity using NanoDrop. <br><br>
 +
2. Repeat Step 2 ~ 5 as from Case I<br>
 +
 
 +
 
 +
[[Image:Mindmap_expt.JPG|thumb|800px|center|Summary of Digestion and Ligation Procedures]]
 +
 
 +
=='''OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2'''==
 +
1) One colony of LuxS(-) W3110 strain containing pLsrA-lysis plasmid was inoculated overnight in 50ml LB inside 250ml flask at 37 degrees C and 225rpm. <br><br>
 +
2) After 16 hour inoculation, 0.5 ml of cell sample was diluted in 50ml fresh LB.<br><br>
 +
3) Diluted cell sample was distributed into the wells of the black-96-well microplate with an amount of 200 µL per well. <br><br>
 +
4) 50µl AI-2-containing supernatant was added into corresponding wells initially (set 1) and 1 hour after incubated inside absorbance-meter (set 2). <br><br>
 +
5) Five different supernatant solutions used, which correspond to the time points when they had been obtained during AI-2 preparation: 3, 4, 5, 6, 8 hours.<br><br>
 +
6) Control samples included
 +
  i) cell suspension alone
 +
  ii) cell suspension with 50µl LB added initially (set 1)
 +
  iii) cell suspension with 50µl LB after 1 hour incubation (set 2)
 +
  iv) cell suspension with 50µl water added initially (set 1)
 +
  v) cell suspension with 50µl water added after 1 hour incubation (set 2)
 +
<br>
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<br><br>
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Latest revision as of 05:47, 27 October 2008



Contents

Protocols

Creation of New Biobrick parts

To create a new biobrick part, the first step is to identify the gene of interest. In this project, our genes of interest are the E7 production gene, Immunity gene, and Lysis gene that is obtainable from the plasmid ColE7-K317 in BW Escherichia Coli strain. Another gene of interest is the LsrA promoter that is obtained from MG1655 Escherichia Coli strain. Information on the genome of interests is extracted from relevant publications and websites such as NCBI net.

For example the gene of interest – Lysis gene:
1. A pair of DNA primers, 10 ~ 15 nucleotides in length are designed and synthesized to flank the forward 3' – 5' end and reverse 5' – 3' end of the gene of interests. The primers also contain blunt ends of the EcoRI, XbaI, SpeI & PstI restriction sites. These primers will hook on to the double strand DNA by complementary pairing and serve as initiation and termination site of DNA amplification.

2. Amplification of the lysis gene occurs by PCR amplification. A gel electrophoresis is run, followed by a gel extraction of the specific interest.

3. The lysis gene is then purified using the PCR purification kit. Purified DNA is tested for concentration and purity using NanoDrop.

4. Next, the purified DNA is digested using EcoRI and PstI restriction enzymes to generate a standard Biobrick part with the gene sequence:

EcoRI – XbaI - Lysis gene – SpeI – PstI

Creation of New BioBricks

Digestion and Ligation of NTU@iGEM System

To ligate 2 Biobrick parts together, firstly we identify one part to be "vector" and the second part to be "insert". There are 2 possibility of ligation:

  • Case I: Insert in front – Vector behind
  • Case II: Vector in front – Insert behind

Case I: Insert in front – Vector behind

1.Digest Insert gene with EcoRI & SpeI and Vector gene with SpeI & PstI.
Purify the digested products with PCR Purification Kit and test for concentration and purity using NanoDrop.

2.Perform a gel electrophoresis of the purified products, followed by a gel extraction using PCR Gel Extraction Kit.

3.Fuse the Insert gene to the Vector gene to form a circular plasmid DNA using T4 DNA Quick Ligase.
Transform the DNA plasmid into Top 10 Competent Cells and select using appropriate antibiotic treatment.
Vector gene contain antibiotic resistant gene that confers resistance to the cells that are successfully transformed.

4.Extract the ligated DNA using MiniPrep Kit and digest the resultant DNA using EcoRI & PstI.

5.Lastly, perform a gel electrophoresis for confirmation. Check if the nucleotide length from the gel run matches the hypothetical nucleotide length of the digested product. If the result matches, ligation is successful.

Case II: Vector in front – Insert behind

1. Digest Insert gene with XbaI & PstI and Vector gene with EcoRI & XbaI. Purify the digested products with PCR Purification Kit and test for concentration and purity using NanoDrop.

2. Repeat Step 2 ~ 5 as from Case I


Summary of Digestion and Ligation Procedures

OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2

1) One colony of LuxS(-) W3110 strain containing pLsrA-lysis plasmid was inoculated overnight in 50ml LB inside 250ml flask at 37 degrees C and 225rpm.

2) After 16 hour inoculation, 0.5 ml of cell sample was diluted in 50ml fresh LB.

3) Diluted cell sample was distributed into the wells of the black-96-well microplate with an amount of 200 µL per well.

4) 50µl AI-2-containing supernatant was added into corresponding wells initially (set 1) and 1 hour after incubated inside absorbance-meter (set 2).

5) Five different supernatant solutions used, which correspond to the time points when they had been obtained during AI-2 preparation: 3, 4, 5, 6, 8 hours.

6) Control samples included 

  i) cell suspension alone
  ii) cell suspension with 50µl LB added initially (set 1)
  iii) cell suspension with 50µl LB after 1 hour incubation (set 2)
  iv) cell suspension with 50µl water added initially (set 1) 
  v) cell suspension with 50µl water added after 1 hour incubation (set 2)




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