What is the purpose of transformation solution in pGLO lab?
In this lab, you’ll transform E. coli cells with the plasmid pGLO, which contains a gene for green fluorescent protein (GFP). This will allow you to grow bacterial colonies that are bright, fluorescent green.
Why did the LB amp ARA glow?
+pGLO, LB/AMP/ARA: They were exposed to AMP and arabinose sugar, causing bacteria to grow with ampicillin resistance. Colonies glowed due to exposure to arabinose sugar and UV light.
Why does pGLO glow in arabinose?
In the pGLO plasmid DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene that codes for GFP. In the presence of arabinose, the GFP gene is turned on, and the bacteria glow brilliant green when exposed to UV light.
What is the role of arabinose in the transformation lab?
The pGLO system incorporates an arabinose promoter which precisely controls expression of the GFP in transformed cells. Expression of the GFP gene can be turned on simply by including arabinose (a sugar) in the growth medium.
What factors might influence transformation efficiency?
The factors that affect transformation efficiency are the strain of bacteria, the bacterial colony’s phase of growth, the composition of the transformation mixture, and the size and state of the foreign DNA.
Why is ampicillin used in pGLO lab?
Antibiotic Selection The pGLO plasmid, which contains the GFP gene, also contains the gene for beta- lactamase, which provides resistance to the antibiotic ampicillin, a member of the penicillin family. The beta-lactamase protein is produced and secreted by bacteria that contain the plasmid.
What is the link between pGLO and ampicillin resistance?
The pGLO plasmid also contains a gene for ampicillin resistance so that successful transformants can be distinguished from cells that have not taken up the plasmid DNA by their ability to grow on a medium containing this antibiotic.
Why is ampicillin added to some plates?
It is added to the culture for the best survival of culturing cells during our experiment. If your vector has ampR gene that codes for b-lactamase, then you’d add ampicillin to screen positives. Other reason is, amp is a broad range bacteriostatic antibiotic, which discourages contaminating bacteria from growing.
What is the purpose of the ampicillin in our agar plates?
Ampicillin is an antibiotic used to selectively eliminate bacteria that have not been transformed with plasmids containing an ampicillin resistance gene.
What are some reasons why transformation may not succeed?
There are a handful of common mistakes that can happen during the transformation process.
- Incorrect antibiotic. Double-check that you are plating on the correct antibiotic.
- Incorrect concentration of antibiotic.
- Excessive freeze-thaw.
- Low amount of DNA transformed.
- Heat shock.
- Recovery Time.
How do you calculate transformation rate?
Usually people calculate the transformation efficiency (%) as: Transformation efficiency (%)= (Total number of PCR positive plants / Total number of inoculated callus) ×100.
What two traits are found on the pGLO plasmid?
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably the green fluorescent protein (GFP) and the ampicillin resistance gene.
Why is arabinose needed for pGLO?
When arabinose is added to E. coli cells that contain pGLO, the GFP gene will be highly expressed, becoming one of the most abundant proteins in the cell. When arabinose is not present, the amount of GFP expression will be close to zero.
What are the 3 genes of interest on the pGLO plasmid?
The pGLO plasmid contains both the promoter (pBAD) and araC gene, but araB, araA, and araD have been replaced by the single gene that codes for GFP, which serves as a reporter gene.
Why are pGLO tubes placed on ice?
The holes poked to allow the DNA in leaves the bacteria leaky. If we don’t keep them on ice, they’ll ‘bleed’ to death. 4. Put tubes directly from ice into 42°C water bath for 50 seconds.
Why is ice cold CaCl2 used in bacterial transformation?
The ice-cold CaCl2 solution facilitates binding of DNA to the surface of the cell, which then enters the cell after a short period of heat- shock (3). Cells that are successfully transformed are usually identified by selection or screening markers such as drug resistance or fluorescence (4).
What is the purpose of the LB amp ARA plate (+) plasmid?
If there are any genetically transformed bacterial cells they would be located on the +pGLO LB/amp and the +pGLO LB/amp/ara plates. This is because both these plates have the plasmid inside them which allows for the transformation. You just studied 16 terms!
How many terms are in the pGLO transformation lab?
pGLO Lab Analysis 16 terms gracebronner Bacterial Transformation Lab: pGLO 34 terms mocha_dog529 Bio Lab Midterm 88 terms aya2 Sets with similar terms pGLO Transformation 19 terms Ayah_Rasheed Biomed Unit 7 57 terms Angela_Silva32 Bacterial Transformation Lab 16 terms GermaineRicablanca Microbiology Lab Exam 115 terms BrittB95
What is pGLO lab analysis?
pGLO Lab Analysis. Plasmids are pieces of circular DNA that are in bacteria which can code for genes that can cause the bacteria to have extra “features”. With the pGLO plasmid this extra “feature” causes bacteria to glow (from the Green Fluorescent Protein that was inserted) and also has resistance to ampicillin (from the beta-lactamase protein).
What is the difference between cells treated with DNA and pGLO?
compared. Cells which were not treated with DNA (-pGLO) should not be expressing the ampicillin resistance gene and will not grow on the LB/amp plates. Cells which were treated with DNA (+pGLO) should contain the pGLO
How does the plasmid pGLO work?
pGLO: How the plasmid works. In this lab, you’ll transform E. coli cells with the plasmid pGLO, which contains a gene for green fluorescent protein (GFP). This will allow you to grow bacterial colonies that are bright, fluorescent green.