A marvelous creation of Biotechnology

1. GLOWING
PLANTS
By: Sakshi Shiram

2. WHY DO WE NEED
A GLOWING
PLANT?
Development of glowing plant can be used to
replace street lights, reducing CO2 emission by
not requiring electricity. Would be a very first
step in creating natural lighting.
The effort also reflects a ‘DIY biology’
movement that seeks to make biotechnology
more accessible to the public.

3. WHAT IS
BIOLUMINESCENCE?
• Bioluminescence is the production and emission
of light by a living organism. Occurs widely in in
marine vertebrate and invertebrates, as well as in
some fungi, microorganisms including some
bioluminescent bacteria and terrestrial
invertebrates such as fireflies.
• Fireflies produce a chemical reaction inside their
bodies that allows them to light up. this type of
light production is called bioluminescence. When
O2 combines with calcium, ATP and the chemical
luciferin in the presence of luciferase, a
bioluminescent enzyme, light is produced.

4. GOING WITH THE GLOW:
Fauna that exhibit
bioluminescence
• Occurs widely among animals,
especially in the open sea,
including fish, jellyfish,
crustaceans and Cephalopod
molluscs
• Fungi and Bacteria
• Invertebrates including
insects: Fireflies and glowing
worms

5. Luciferin and luciferases
Luciferin is a complex molecule
that is similar to Chlorophyll. The
bioluminescent light that is emitted
when the luciferin is oxidized is a
byproduct of the reaction. Also the
light is called ‘cold light’ because
there is almost no heat that
accompanies the emission of light.
The bacterial luciferin- luciferase
system is encoded by a set of genes
labelled the lux operon.

7. Lux operon:
 LuxI gene synthesizes auto
inducer which is produced
continuously and it diffuses
across the cell membrane.
 It bounds to LuxR protein
and causes formation of
active dimer molecule
initiating transcription.

8.  Active LuxR- Autoinducer complex is formed.
 RNA Polymerase can bind to the Lux promoter site and
trscribe operon.
 Lux C, LuxD, LuxE are all involved in production of substrate
oxidized by the enzyme luciferase in the production of light.
 Lux A and Lux B are the two subunit of the luciferase enzyme.
 When this joins, the active luciferase enzyme is completed
and bioluminescence commences.

9. pGL2 VECTOR:
Suitable vector: the vector which is
suitable for the cloning of luc gene is
pGL2- control vector.
Restriction enzymes:
 HindIII
 BamHI
 SaII
 Xhol
 BglIII
 KnpI

10. Cloning process:
 Before cloning, firstly the pGL2, control vector is designed in
which the luc gene is present.
 Then the cells of E.coli JM109 re transfected with the pGL2
vector and then cloned
 The transfection of vector into E. coli may be mediated by
calcium phosphate, or electroporation.
 pGL2 vector contains the SV40 promoter and enhancer
sequences, resulting in strong luc gene expression in the cells.

11.  A complimentary DNA clone of the firefly luciferase gene under the
control of a plant virus promoter (cauliform mosaic virus 35S RNA
promoter) was introduced into plant protoplast cells by
electroporation.
 Another method of introducing the cDNA into the plant is by the use
of Agrobacterium tumefaciens.

12. Agrobacterium tumefaciens?
 The ability of Agrobacterium to transfer genes to plants and fungi is used
in biotechnology, in particular, genetic engineering for plant
improvement.
 A modified Ti or Ri plasmid can be used. The plasmid is 'disarmed' by
deletion of the tumor inducing genes; the only essential parts of the T-
DNA are its two small (25 base pair) border repeats, at least one of which
is needed for plant transformation.
 The genes to be introduced into the plant are cloned into a plant
transformation vector that contains the T-DNA region of the disarmed
plasmid, together with a selectable marker (such as antibiotic resistance)
to enable selection for plants that have been successfully transformed.

13. SHSID_China:
1. Luciferase gene:
Lux operon
2. A new part: LuxG
3. Target
plant: Nicotiana
tabacum
Transforming plasmids
obtained into
agrobacterium and get
agrobacterium
containing the target
plasmids.
5. Plasmid for
agrobacterium-
mediated
transformation-pHB
4. Gene Transfer Method:
Agrobacterium-mediated
transformation
6. Agrobacterium-
mediated
Transformation
7. Observation

14. PUBLICATION 1:
Illustrated the use of luxA and luxB as reporter
gene required fusion of both genes in eukaryotic
system.
Reported the use of more sensitive reporter gene
other than the lux genes for efficient gene
transfer into the plant cells.
They highlighted the mysterious solution for the
above question. Introduction of luc cDNA into
Cauliform mosaic virus 35s RNA promoter. To
ensure the productive results, a 3 prime end
fragment from the nopaline synthase (nos) gene
was fused behind the luc cDNA as this nos 3
prime end fragment contains a polyA signal
known to be functional in plant cells. The entire
gene was inserted into the plasmid vector pUC19

15. PUBLICATION 2:
San Francisco-based entrepreneur Antony Evans plans to insert genes
from bioluminescent bacteria into a species of flora as a first step to
creating glowing trees. (Antony Evans). Evans and his colleagues,
biologists Omri Amirav-Drory and Kyle Taylor, want to create plants that
literally glow.
They started with a software called GENOME COMPILER, which proved
helpful to look up the Vibrio fischeri genes, and then we do something
called code and optimization, which basically adjusts the sequences so
that they [work] in plants instead of in bacteria. Then synthesize the DNA
and print the DNA. The printed DNA form will be sent to the DNA making
company.
After getting the resulting DNA, it is inserted into the bacterium called
Agrobacterium. That bacterium is very clever, it has figured out how to do
genetic engineering on its own. [The bacterium] inserts the DNA into the
female gametes of the plant. We can grow the seeds that come from those
flowers, and we’ll have the DNA that we designed on the computer in the
plant.

16. Publication 3: Variable Patterns of
Expression of Luciferase in Transgenic
Tobacco Leaves
A carboxyl-terminally modified firefly luciferase, encoded as a gene fusion to the
neomycin phosphotransferase gene (which confers kanamycin resistance), was
found to be enzymatically active for both enzymes when expressed in bacteria and in
transgenic plants. Transgenic tobacco plants which expressed luciferase uniformly in
all areas of the leaf, and assays for luciferin, demonstrated that luciferin rapidly
penetrates all regions of a tobacco leaf in at least two dimensions. Depending on the
test gene structure or, presumably, on the transferred DNA (T-DNA) insertional
context, other transgenic plants were obtained that expressed luciferase with a wide
range of nonuniform patterns from nominally the same cauliflower mosaic virus 35S
promoter. For instance, the veins can be dark, while only the interveinal regions of
the leaf lamina glow, or only the small capillary veins glow, or only the major veins
glow. Local and/or systemic induction in response to wounding was also
demonstrated.

17. References:
 https://www.smithsonianmag.com/innovation/creating-a-new-
kind-of-night-light-glow-in-the-dark-trees-9600277/
 https://www.jstor.org/stable/2355785?seq=1#page_scan_tab_co
ntents
 http://2018.igem.org/Team:SHSID_China/Design
 http://archive.bio.ed.ac.uk/jdeacon/microbes/crown.htm
 https://www.promega.in/products/luciferase-assays/genetic-
reporter-vectors-and-cell-lines/pgl2-luciferase-reporter-
vectors/?catNum=E1641
 http://news.mit.edu/2017/engineers-create-nanobionic-plants-
that-glow-1213