Molecular biology of the cell with Lab

Instructor: Assem Duisembekova

Section #1
Week #1
Title: Genetic transformation of bacteria with a gene for Green
Fluorescent Protein (GFP)
Name: Alibek Ysmaiyl
Introduction

In general, genetic transformation is defined as a process by which the genetic material

carried by bacterial cell is altered by the introduction of foreign DNA into its genome. Moreover,
the genetic transformation was firstly discovered by Griffith in 1928 by transformation in
Streptococcus pneumoniae, and demonstration of DNA as the transforming material was
discovered by Avery in 1944 [1].

In this experiment, bacteria called E.coli will uptake a gene which encodes for Green
fluorescent Protein (GFP). Under ultraviolet light, bacteria with GFP gene will demonstrate a
brilliant green color.

Figure 1.The structure of pGLO plasmid [2]

Broadly, plasmid DNA contains one or more traits other than GFP gene which can be helpful in
survival. In this laboratory procedure, pGLO has beta-lactamase gene which provides the
resistance for ampicillin antibiotic (figure 1) [2].

The main purpose of this lab is to learn about the genetic transformation from one organism
to another one. The objectives are to perform the genetic transformation procedure into E.coli as
well as to determine the degree of efficiency (transformation efficiency) of genetically altered
organism.

The general hypothesis is that E.coli bacterial cells will achieve a trait if the plasmid which
carries the gene that codes for GFP is transformed into bacterial cells. In order to receive the
properly stated results, the hypothesis is made for each prepared plate. So, there will be growth
of bacteria that does not demonstrate the GFP gene in the plate containing +pGLO LB/amp.
However, +pGLO LB/amp/ara plate will grow bacteria which shows the GFP gene. The pGLO
LB/amp/ara will not show the growth of bacteria, whereas pGLO LB will contain bacteria with
no GFP gene. The main point of these hypotheses is that GFP switches on in transformed cells in
the presence of arabinose (ara) sugar in the nutrient medium.
Materials and method

Materials: sterile 1.5 mL microtubes, sharpie pen, sterile transfer pipets, pipettor, sterile loops,
ice basket, LB plate, 2 LB/amp plates, water bath at 42 0C, sterile LB broth, 370C incubator,
pGLO plasmid DNA, sterile CaCl2 transformation solution, foam microtube rack.

Procedure

After labelling two micro test tubes +pGLO and pGLO respectively, 250l of
transformation solution was pipetted into each tube.

Single colony was picked up from the starter plate containing E.coli with sterile loop, and
transferred into +pGLO tube with transformation solution. This was repeated for pGLO
tube with new sterile loop.

10l of pGLO plasmid was added into +pGLO cell suspension tube and both of tubes
were placed into ice for 10 minutes.

After that, both tubes were placed into water bath at 42oC for exactly 45 seconds and then
put into ice for 2 minutes (heat shock).

250l of Luria-Bertani (LB) nutrient broth was added to each tube in order to recover
after heat shock.

Both tubes were placed in incubator at 370C for one hour.

Then, 100l from each tube was pipetted into corresponding plates with new sterile
pipette in each case.

Finally, the suspensions were spread on the surface of plates and plates were incubated at
37oC.

Results

Table 1.Observations on growth of +pGLO and pGLO bacteria on different 4 plates

General Bacteria Colour

growth number visible
under UV
light
LB agar pGLO Positive TNTC -
LB/amp pGLO Negative 0 -

LB/amp +pGLO Positive 12 -

LB/amp/ara +pGLO Positive 6 Green

*TNTC too numerous to count

Calculations:

In order to derive transformation efficiency, which means how efficient cell can uptake
extracellular DNA and express it, firstly total amount of DNA is calculated:

DNA (g) = (concentration of DNA (g/l) x (volume of DNA in l), where concentration =
16 ng/l=0.16 g/l and volume = 10 l

DNA (g) = 0.16 * 10=1.6 g

Volume on LB ara plate

Fraction of DNA used for LB/ara plate for +pGLO bacteria = total volume =

100/510 = 0.196

pGLO DNA spread (g) = total amount of DNA * fraction of DNA = 0.196 * 1.6 g = 0.313
g

Transformation efficiency = Total number of cells growing on LB/ara agar plate / Amount of
DNA spread on the agar plate = 12/0.313 g= 38.3 transformants/g

Discussion

This lab was successfully performed and the hypotheses are supported by obtained results. It
is clearly seen that pGLO LB/amp plate did not show any bacterial growth and the reason for
this is that bacterial cells on this plate were not transformed with pGLO which means that there
was no ampicillin resistance gene. Therefore, in general there was no growth of bacteria on
pGLO LB/amp plate. Moreover, the interesting point of results is not only the growth of bacterial
cell on +pGLO LB/amp/ara plate, but also the glowing under the UV light. This glowing
suggests the presence of GFP gene. In addition to this, only this plate contained the arabinose
sugar which is necessary for transcription of GFP gene. The +pGLO LB/amp plate have also
shown the bacterial growth. Transformation efficiency for this was calculated and it is 38.3
transformants/g.
Reference list:

1. Lorenz, M. G., & Wackernagel, W. (1994). Bacterial gene transfer by natural genetic transformation in
the environment. Microbiological Reviews, 58(3), 563602.

2. Blaber, M. Genetic Transformation (using bacteria and the pGLO plasmid)

http://www.mikeblaber.org/oldwine/BCH4053l/Lecture07/Lecture07.htm (accessed Jan 29, 2017).