Group I.

1/ HOW THE PATHOGENS PROTEIN IS TAKEN INTO CIRCULATING SYSTEM?

The antigens in transgenic plants are delivered through bio-encapsulation, i.e, the tough outer wall of
plant cells, which protects them from gastric secretions in stomach and finally break up in the intestines.
The antigens are released, and these proteins are able to bind specifically to various glycolipids and
glycoproteins located on the surface of the cells of the intestinal mucosa and somehow to trigger these
cells to internalize the antigens and to transport them to the circulation. Finally they are taken up by M
cells in the intestinal lining that overlie peyer's patches and gut-associated lymphoid tissue (GALT),
passed on to macrophages, other antigen-presenting cells; and local lymphocyte populations, generating
serum IgG, IgE responses, local IgA response and memory cells, which would promptly neutralize the
attack by the real infectious agent.
REMEMBER STOMACH IS NOT A PLACE FOR ABSORBING BUT GRINDING FOOD!

2/ HOW TO MAKE VIRULENCE GENE EXPRESS IN EDIBLE PART
We should use a specific plasmid which integrate the desired gene into the specific site of the operon
that likely express in the edible portion only. How to recognize those operons? Extract the total mRNA
which express in the banana, then conduct a large scale of pyro-sequencing which result in total genes
will be transcribed just in the banana only (not in leaf and other parts). Using BLAST to track against
these genes on the GenBank and identify the potential operon which will express together. Make a
plasmid carrying desired gene (cholera) and use mutagenesis knock-in technique to introduce that
plasmid into the banana callus. The transferred plasmid just can be integrated into the prior-targeted
operon which will express in banana only.
3/ How to make FIRST AND SECONDARY ANTIBIOTICS
primary antibody
This description assumes you have available purified protein. Run the protein on an SDS-PAGE gel.
Stain the gel with KCl. The KCl forms a precipitate with the SDS. Since the area with the protein has a
low concentration of SDS, the area with the protein will not show a precipitate. This will allow you to see
the protein band as a clear band against a milky white precipitate on the rest of the gel. Carefully cut out
the band and soak it in 1 mL PBS buffer. Crush it and make an emulsion with 1 mL Freund's Complete
Adjuvant (which is an oily substance). The complete adjuvant contains microbacteria (an immune
stimulant) to increase the immune response. Inject this subscapularly into a rabbit. This is your first
inoculation. Only use the complete adjuvant for the first inoculation. NEVER inject a rabbit with complete
adjuvant more than one time. Rest the rabbit for one month, then repeat the process using an
incomplete adjuvant. You can expect to see good antibody titers about 10 days after the second booster.
Bleed the rabbit. You can expect about 30 to 40 mL per bleeding, and about 50 percent of the volume is
serum. Now you have rabbit antisera. To get your primary antibody, dilute the rabbit antisera in blotto
(aka Carnation Nonfat Dry Instant Milk) and apply it to your nitrocellulose blot. Make sure you dilute
1:500 to 1:100 in blotto; less dilution will give you background binding and really muddy up your results.


Secondary antibody
Grab a catalogue and look for a goat-anti-rabbit antibody conjugated to horseradish peroxidase (HRP).
The goat-anti-rabbit is your secondary antibody (the one that "sticks" to the primary antibody) and the
HRP is the conjugated enzyme that will allow you to visualize your protein.
4/Why do we need to use DH5 instead of using other E.coli strains?
Because, the main role of DH5 is cloning application so that it has several features that make useful for
recombinant DNA methods .For example, it contains recA1 and endA1 which increase insert stability
and improve the quality of plasmid DNA prepared form minipreps
5/ Why do we need 2 plasmid pBluescript and pCAMBIA instead of just pCAMBIA?
Because pBlusescrip is used for cloning, it contains a multiple cloning site sequence (MCS), antibiotic
resistance sequence to ampicillin and an E.coli and f1 helper phage origin of replication. On the other
hand, pCAMBIA is an expression cassette which contains a lot of region for transformation. With
pCAMBIA we can observe the result clearly.
Group II
Microbial production of short-chain alkanes
1. Figure explanations.
This figure aims to check only the produced free fatty acid under the activity of modified thioesterases
and TesA(L109A). Therefore, the FadD gene was deleted (the pathway will end at FFAs). There are
some abbreviations that need to be considered:
.coli


a. The figure compares the net production of fatty acid between 4 types of gene:
TesA, TesA (L109P), UcfatB and TesB in the fadD-deleted W3110 strain together with the control strain
harboring an empty vector. The result shows that TesA and TesA(L109P) have the highest efficiency.
b. Distribution of FFAs produced in thefadD-deleted W3110 strain overexpressing TesA (hatched bar)
and TesA(L109P) (solid bar). The percentage ratios of FFAs produced are also shown. The figure
shows that TesA(L109P) produces mainly C14, which is shorter as compare to the C16 in TesAs
products. As the study aims to product Short chain alkanes (C9-C14), the TesA(L109P) is used for
further analysis.
c. The chain length distribution and percentage ratios of FFAs produced in fadDdeleted
GAS1 (c) and fadD-deleted GAS2 (d) expressing TesA(L109P) are shown. After deleted FadE and
FadR, the ratio of C10 free fatty acid increases significantly because of the enhancement in fabH.

2. Why use 31oC in culturing?
Ans:
- They prefer to live at a higher temperature rather than the cooler temperatures. E.coli is a Gram-
negative organism that cannot sporulate. Therefore, it is easy to eradicate by simple boiling or basic
sterilization.
- The expression level of CER1 was found to be higher at 30uC, compared with results obtained at other
temperatures examined (Supplementary Fig. 10); therefore fedbatch fermentation was performed at 30
census degree.

3. Why included Benzoic acid in the medium?
Anw:
- The benzoic acid replaces at least 20% of the fatty acid. Smaller amounts of benzoic acid do not
noticeably improve the results. Much larger amounts of benzoic acid may
be used. While both fatty acid and benzoic acid should be used to drying , a very high percentage of the
fatty acid may be replaced. For example, or more, of the fatty acid may be replaced with benzoic acid,
the upper limit being governed by the desired air drying properties.
- Purpose: that the improved properties of the present alkyds result from a reaction between the benzoic
acid and the fattyacid.
Extra question: We can replace acid benzoic by other compound?
Anw: No.
- Explanation: While some reaction apparently results between fatty acids such as lauric and stearic
acids, the reaction proceeds more slowly and to a more limited extent.
4. Why 2 enzymes in ligation?
Anw:
- Usually, scientists select two different enzymes for adding an insert into a vector (one enzyme on the 5'
end and a different enzyme on the 3' end). This ensures that the insert will be added in the correct
orientation and prevents the vector from ligating to itself during the ligation process. If the sticky ends on
either side of the vector are compatible with each other, the vector is much more likely to ligate to itself
rather than to the desired insert. If you are in this situation, it is important to treat the digested vector
backbone with a phosphatase before performing the ligation reaction (phosphatase removes the 5'
phosphate and therefore prevents the ligase from being able to fuse the two ends of the vector together).
5. Why we need to combine plasmids?
- The reason why we need to combine is to make sure that the 2 different genes are successfully
transferred into the E coli.
- If we use 2 plasmids separately the following result can happen



- Thus, the combination is just to increase the presence of both of the genes
probability.
Group III

1. How many products from TPCR ?

- 3 products ( complete plasmid, TPCR intermediate product, incomplete plasmid, why incomplete ? )

2. How can we get 1 product ? ( gene only, vector only ? )
If we have short time elongation => get gene
Step 1: denature both vector
Step 2: 60oc is good for primer
1.5 to amplify 1 gene only
5. Why 13 cycles ?
- To get enough product to be a primer
Step 1 , step 2: to make enough product to be a primer for step 3


Group IV
Loop-mediated Isothermal Amplification

1. Disadvantage of LAMP PCR?

In LAMP PCR, we cannot get the purified prodcuts (smear band) because the prodcuts produced have
zigzag form.

2. How many steps are there in LAMP PCR?

There are 2 main steps: annealing and extension, inactivating enzymes.
3. Why dont we use the denaturing step in LAMP assay?

As double stranded DNA is in the condition of dynamic equilibrium at the temperature around 65C, one
of the LAMP primers can anneal to the complimentary sequence of double stranded target DNA, then
initiates DNA synthesis using the DNA polymerase with strand displacement activity, displacing and
releasing a single stranded DNA. Therefore, there is no need for heat denaturation of the double
stranded DNA into a single strand.

4. Why do we use different concentration/ amount of primers?
LAMP is performed for 60 min at 65 C in 25 L of a mixture containing 2 L of extracted DNA, 40 pmol
each of FIP and BIP, 5 pmol each of F3 and B3. There is the difference in the amount of primers used
because FIP and BIP are used over the steps while F3 and B3 are just involved in only 1 step.
Group V
advantages of iPSCs compared to other stem cells are
3. Why do we need to use iPSCs?
We use iPSCs because of these reasons:
a) iPSCs can be created from the tissue of the same patient that will receive the transplantation, thus
avoiding immune rejection
b) the lack of ethical implications because cells are harvested from a willing adult without harming
them.
c)These patient-specific cells can be used to study diseases in vitro, to test drugs on a human model
without endangering anyone, and to hopefully act as tissue replacement for diseased and damaged
cells.
2. Explain simple sequence lenght polymorphism.
Simple Sequence Length Polymorphisms (SSLPs) are used as genetic markers with Polymerase Chain
Reaction (PCR). An SSLP is a type of polymorphism: a difference in DNA sequence amongst
individuals. SSLPs are repeated sequences over varying base lengths in intergenic regions of
deoxyribonucleic acid (DNA). Variance in the length of SSLPs can be used to understand genetic
variance between two individuals in a certain species.
1. Why the chromosome number in the end of embryo is 4n? What is its function?
The chromosome in the blastocyst is 4n because we use the technique call electro fusion to fuse the 2
cell-embryo into 1 cell-embryo and that embryo has 4n. If we injected the iPSCs (2n) into the normal
blastocyst (2n), we just have a chimeric animal with the DNA from normal blastocyst and our iPSCs. And
if we used tetraploid complementary blastocyst (4n) and injected iPSCs into this blastocyst, we will have
a 100% animal with the DNA from the iPSCs and the blastocyst will become the placenta.
The function of this tetraploid complementary blastocyst (4n) is that :
If we use iPCSs to make a clone animal, the placenta of this animal is very weak so the baby will
die early. So we use tetraploid to make placenta of this clone animal stronger, that is mean the baby will
have the chance to survive.
And this 4n will make the implantation of blastocyst into the uterus stronger and this lead to the
percent of cloning animal will increase.
The mechanism of tetraploid complementary is still unknown, results are collected from experiment.
Group VI
1. Why do we have to use 2 kinds of enzymes?
Almost all restriction enzymes have different restriction sites. Both ends of the vector must
complementary both ends of the enzymes.
To be able to clone a DNA insert into a cloning or expression vector, both have to be treated with two
restriction enzymes that create compatible ends. At least one of the enzymes used should be a sticky
end cutter to ensure that the insert is incorporated in the right orientation.
Sticky end cutters are the enzymes cut both strand of the target DNA at different spots creating 3'- or 5'-
overhangs of 1 to 4 nucleotides (so-called sticky ends).
2. Why do we have to use 2 kinds of E.coli?
E.coli Dh5-alpha: used to clone the gene with higher success.
E.coli BL21: high-level protein expression.
We must use E.coli DH5alpha first to confirm that this gene is cloned.
3. How do we purify the PCRs products?
After running PCR, we can get many proteins. To get the right product, we need to design His-tag
sequence on the primers that the molecule tags with histidine at histdine site.
4. What is hbs gene?
hbs gene the gene encode for HBsu protein. This protein is the homolog of HU protein - a type of
histone-like protein in E.coli. HBsu binds non-specifically to DNA to stabilize the secondary structure of
DNA. It's also a heat stable protein that helps bacteria survive in high temperature.

Group VII
1. How does T4 DNA polymerase work?

Answer: T4 DNA polymerase is the enzyme that express two activities: polymerase on 5-3 direction and
exonuclease activities on 3-5direction. Two activities can occur at the same time. In the presence of
dNTP, the polymerase activities is dominant because it has resource (dNTP) to work. In the absence of
dNTP, exonuclease (elimination of nucleotide) is dominant. In our experiment with digested plasmid (
has sticky ends), we use T4 in the presence of dGTP, so that the enzyme will express the exonuclease
activities on 3 end of plasmid. In the presence of only dGTP, the polymerase activity can not work (lack
of the nucleotide resource to bind to 5 end) until the enzyme reach first cytosine, guanine can bind to,
on the 5 strain (correspond to guanine on 3 strain. The result is the plasmid vector with long 5
overhang on the ends

2. The principle of ligation independent cloning?

Answer: Ligation-independent cloning (LIC) is the technique that creates the long and compatible ends
between plasmid vector and the gene to be inserted. The long overhang on sticky end help two fragment
to easily combine to each other due to a lot of hydrogen bonds formed along the strain. Advantages: 1)
We dont have to use ligase enzyme to combine two fragment and 2) The efficience is higher than the
process relied on ligase because plasmid and gene bind strongly to each other


3. Why do we create 4 LIC sites instead of 2 LIC sites on the plasmid?

Answer: To compare the efficiency between many approach. Its does not matter if we use just two LIC
sites
Group VIII
ENHANCEMENT OF NUTRITIONAL QUALITY OF WHEAT BY METABOLIC ENGINEERING OF
ISOFLAVONE PATHWAY
Question 1: Why do we use two promoter? Which one has the better activity?
-Two promoters are used: 35S derived from the cauliflower mosaic virus and oleocin derived from the
soybean. We used 2 promoters for the comparison of the activity to express our desired gene.
-According to the result of the HPLC, it showed that the Genistein concentration and Genistein range of
O-IFS is higher than that of 35S-IFS, thus the oleocin promoter has work more effective than 35S
promoter.



Question 2: what are the binary vectors?
-The binary vectors are the combination of two or more vectors in the transformation. In this article, one
vector contain our desired gene IFS, one vector contains the transcription factor CRC gene which
enhance the expression of our desired gene and other gene in the vectors, and the last one contain the
bar gene as selectable marker.

Question 3: Can we use other test to check the present of our desired gene in the transgenic
plant?
-Yes, we can. - We can a use the Western blot to detect the fluorescence and chemi-luminescence or
we can use GUS gene to detect our desired gene because Gus encodes for -glucuronidase which
make the D-glucuronic acid become blue substance so that it makes the transgenic plants turn blue.