Berkeley LBL/Methods

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|[[Berkeley_LBL/Methods|Methods]]
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|[[Berkeley_LBL/Notebook|Notebook]]
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|[[Berkeley_LBL/Results|Results and Discussion]]
|[[Berkeley_LBL/Resources|Resources]]
|[[Berkeley_LBL/Resources|Resources]]
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==Experimental==
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=== Subcloning ===
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The genes of interest from ''Rhodobacter sphaeroides'', ''Synechocystis sp'', and ''Heliobacillus mobilis'' were amplified using PCR (the details of which can be found in Protocols).  The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into ''E.Coli'' (either DH10B or NovaBlue) and glycerol stocks for these cells were saved.
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=== Constructs ===
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The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.
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'''Magnesium-Chelatase'''<br>
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''Rhodobacter sphaeroides''
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{| border="1"
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|-
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!Construct Name
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!Construct Details
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|-
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|pET3a-R-bchH
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|T7p-rbs-bchH-T7Term
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|-
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|pET3a-R-bchI
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|T7p-rbs-bchI-T7Term
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|-
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|pET3a-R-bchD
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|T7p-rbs-bchD-T7Term
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|-
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|pET3a-R-bchHID
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|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term
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|}
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''Synechocystis sp.''
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{| border="1"
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|-
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!Construct Name
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!Construct Details
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|-
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|pET3a-S-chlH
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|T7p-rbs-chlH-T7Term
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|-
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|pET3a-S-chlI
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|T7p-rbs-chlI-T7Term
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|-
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|pET3a-S-chlD
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|T7p-rbs-chlD-T7Term
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|-
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|pET3a-S-chlHID
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|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term
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|}
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'''Enzymes for reactions after Mg-insertion step'''<br>
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''Heliobacillus mobilis'' (T7 expression vector pET29bEBBX was used for subcloning of heliobacteria)
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{| border="1"
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|-
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!Construct Name
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!Construct Details
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|-
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|pET29-bchB
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|T7p-rbs-bchB-T7Term
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|-
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|pET29-bchE
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|T7p-rbs-bchE-T7Term
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|-
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|pET29-bchM
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|T7p-rbs-bchM-T7Term
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|-
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|pET29-bchN
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|T7p-rbs-bchN-T7Term
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|-
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|pET29-bchI
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|T7p-rbs-bchI-T7Term
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|-
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|pET29-bchL
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|T7p-rbs-bchL-T7Term
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|}
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=== Expression ===
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Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel. (all expressions were done by Joyce, Mimi, and Konniam; gel was done on 10/24/07)
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=== Enzyme Activity Assays ===
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The ''E.Coli'' cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and  fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from ''Rhodobacter sphaeroides'' or ''Synechocystis sp'') is more efficient.
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== '''Subcloning Protocols''' ==
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'''''Polymerase Chain Reaction (PCR) Protocols:'''''
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[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]
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[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]
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'''''Clean Up/ Purification Protocols:'''''
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[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
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[[Berkeley_LBL/GelExtraction|Gel Extraction]]
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'''''DNA Gel Eletrophoresis Protocol:'''''
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[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]
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'''''Restriction Digestion Protocols:'''''
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[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]
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[[Berkeley_LBL/Digestion2|Analytic Digestion]]
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'''''Ligation Protocol:'''''
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[[Berkeley_LBL/Ligation|Ligation]]
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'''''Transformation Protocols:'''''
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[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]
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[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]
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[[Berkeley_LBL/Electroporation|Electroporation Transformation]]
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'''''Miniprep Protocol:'''''
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[[Berkeley_LBL/Miniprep|Miniprep]]
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== '''Expression Protocols''' ==
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'''''Protein Expression Protocols:'''''
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[[Berkeley_LBL/Overexpression|Overexpression]]
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'''''Protein Analysis Protocols:'''''
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[[Berkeley_LBL/Sonication|Sonication]]
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[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]

Latest revision as of 06:51, 27 October 2007

Home Project Description Methods Notebook Results and Discussion Resources


Contents

Experimental

Subcloning

The genes of interest from Rhodobacter sphaeroides, Synechocystis sp, and Heliobacillus mobilis were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into E.Coli (either DH10B or NovaBlue) and glycerol stocks for these cells were saved.

Constructs

The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.

Magnesium-Chelatase
Rhodobacter sphaeroides

Construct Name Construct Details
pET3a-R-bchH T7p-rbs-bchH-T7Term
pET3a-R-bchI T7p-rbs-bchI-T7Term
pET3a-R-bchD T7p-rbs-bchD-T7Term
pET3a-R-bchHID T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term


Synechocystis sp.

Construct Name Construct Details
pET3a-S-chlH T7p-rbs-chlH-T7Term
pET3a-S-chlI T7p-rbs-chlI-T7Term
pET3a-S-chlD T7p-rbs-chlD-T7Term
pET3a-S-chlHID T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term


Enzymes for reactions after Mg-insertion step

Heliobacillus mobilis (T7 expression vector pET29bEBBX was used for subcloning of heliobacteria)

Construct Name Construct Details
pET29-bchB T7p-rbs-bchB-T7Term
pET29-bchE T7p-rbs-bchE-T7Term
pET29-bchM T7p-rbs-bchM-T7Term
pET29-bchN T7p-rbs-bchN-T7Term
pET29-bchI T7p-rbs-bchI-T7Term
pET29-bchL T7p-rbs-bchL-T7Term

Expression

Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel. (all expressions were done by Joyce, Mimi, and Konniam; gel was done on 10/24/07)

Enzyme Activity Assays

The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.

Subcloning Protocols

Polymerase Chain Reaction (PCR) Protocols:

PCR (Using Phusion Polymerase)

PCR (Using TaKaRa Ex Taq Polymerase)

Clean Up/ Purification Protocols:

PCR Clean Up/Purification

Gel Extraction

DNA Gel Eletrophoresis Protocol:

DNA Gel Electrophoresis

Restriction Digestion Protocols:

Digestion for PCR Product or Miniprepped DNA

Analytic Digestion

Ligation Protocol:

Ligation

Transformation Protocols:

KCM Competent Cell Production

KCM Competent Cell Transformation

Electroporation Transformation

Miniprep Protocol:

Miniprep

Expression Protocols

Protein Expression Protocols:

Overexpression

Protein Analysis Protocols:

Sonication

SDS-PAGE

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