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UAS Praktikum Rekayasa Genetika BT5103

Final Exam of Genetic Engineering Practice BT 5103

1.a Sonia menggunakan protocol alkaline lisis untuk ekstraksi plasmid DNA E.coli yang telah
ditransformasi.Untuk pertama kalinya dia meyiapkan bahan-bahan agar menghemat
pengeluaran.. Namun, dia memiliki kesulitan untuk mendapatkan konsentrasi yang baik
dari plasmid DNA-nya, ketika dia melakukan elektroforesis dan visualisasi, dia tidak
melihat satupun pita DNA bahkan pita yang sangat tipis sekalipun. Anehnya adalah ketika
dia mengukur hasil ekstraksi tersebut dengan nanodrop, dia mendapatkan konsentrasi DNA
yang murni (ratio ~ 1,89) sekitar 5,5 ng/ul .Jelaskan mengapa hal tersebut dapat terjadi?

Sonia is using the protocol of alkaline lysis to extract plasmid DNA from transformed
E.coli. This is the first time she prepare the reagents by herself because she can not afford
kits anymore. Anyway, she is having trouble obtaining good concentrations of her plasmid
DNA, because when she ran electrophoresis and visualized she did not see any or a very
weak band of DNA, but the most strange thing is when she quantified her extractions in the
nanodrop, she obtained the concentrations of pure DNA (ratio ~ 1,89) of about 5,5 ng/ul
and she don't really know what is going on?

b. Edo melakukan isolasi DNA plasmid dengan menggunakan metode alkaline lisis untuk
mendapatkan DNA dengan konsentrasi yang tinggi. Dia melakukannya beberapa kali
namun secara tak terduga, dia mendapatkan 2 pita dan bahkan terkadang muncul 4 pita
pada gel elektroforesis seperti pada gambar di bawah ini. Sekarang Edo kebingungan apa
yang terjadi pada DNA plasmid miliknya? Padahal dia mengira hanya ada 1 pita yang akan
muncul

Edo is conducting Plasmid DNA isolation using alkaline lysis method to obtain a large
amount of DNA. He did it several times but unexpectedly on the electrophoresis gel he
obtained two bands and sometimes four bands like this picture below. Now he is so
confused what happens to the plasmid? even though he only thought a band would appear
as he expected.
c. Hanifah telah melakukan ekstraksi DNA plasmid dengan menggunakan alkaline lisis
dengan metode SDS-Midi Prep. Dia melarutkan DNA dengan 100 ul Buffer TE dan
menggunakan sebanyak 7 ul plasmid DNA + 3 ul loading dye pada masing-masing well
pada gel. Dia menjalankan elektroforesis dengan 50 V selama 2.5 jam dan ketika dia
memeriksa pita dengan Gel Doc, hasil menunjukkan adanya pita yang smear. Dapatkah
anda menginterpretasikan hasil dari gel tersebut?
Hanifah extracted plasmid DNA using alkaline lysis with SDS - Midi Prep method. She
dissolved the final DNA in 100ul of TE buffer and loaded 7ul of Plasmid DNA + 3ul of
Loading Dye in each well of Gel. She ran the electrophoresis with 50 V for 2,5 hours and
when she checked for the bands in Gel Doc, it showed smeared bands. She was told that
plasmid DNA should give three bands. She got all the three bands but were smeared. Can
you suggest what can she interpret from the Gel? Can anyone help her in analyzing a
plasmid DNA Gel?

d. Apa yang dapat menyebabkan plasmid DNA dapat terkontaminasi oleh DNA genom atau
RNA serta bagaimana untuk mengatasi masalah tersebut?

What causes plasmid DNA can be contaminated with genomic DNA and/or RNA and how
to overcome those problems?
Problem Possible cause Solution
Plasmid DNA is
contaminated with genomic
DNA
Plasmid DNA is
contaminated with RNA

e. Selama proses isolasi plasmid, bagaimana kita bisa yakin bahwa hanya plasmid DNA yang
telah diisolasi dan tidak ada DNA kromosom?
During the process of plasmid isolation, how can we be sure that only plasmid DNA has
been isolated and there is no chromosomal DNA?
2. Prosedur alkaline lysis untuk ekstraksi plasmid DNA menggunakan 3 solution:
Solution 1 : EDTA, RNAse, Glucose, Tris
Solution 2: SDS, NaOH
Solution 3 : K Acetate

The alkaline lysis procedure for extraction of plasmid DNA uses three solutions:
Solution 1: EDTA, RNAse, Glucose, Tris
Solution 2: SDS, NaOH
Solution 3: K Acetate

a. Apakah fungsi dari masing-masing komponen tersebut?


What is the function of each of the listed components?

b. Apa yang terjadi pada DNA plasmid, DNA kromosom, RNA dan protein pada masing-
masing step?
What happens to plasmid DNA, chromosomal DNA, RNA, and proteins at each of the
steps?

Dinda melakukan isolasi plasmid DNA dari E.coli hasil transformasi dengan menggunakan
metode alkaline lisis dengan sedikit modifikasi (Babitha et al, 2010). Langkah kerjanya
adalah sebagai berikut:
Dinda is conducting Plasmid DNA isolation from E.coli transformed by using alkaline lysis
method with modification (Babitha et al, 2010). The procedure for extraction of plasmid
DNA:
1. About 1.5 mL of bacterial cultures grown in 3ml of L-broth containing appropriate antibiotics
was transferred to microfuge tubes and spun at 12,000xg for 60 seconds to pellet down the
cells.
2. The remaining 1.5ml culture was transferred to the same tube after discarding the
supernatant and centrifuged to collect cells.
3. The supernatant was carefully removed with a fine-tip pipette and the pellet was thoroughly
resuspended in 200ul of Solution I (Sterile distilled water and RNase A solution – (2.5mg/ml),
stored at 4°C) by vortexing.
4. To the reaction mixture, 200µL of Solution II (Alkaline SDS solution – 1% NaOH and 2 % sodium
dodecyl sulfate (SDS), stored at room temperature) was added and the tube was gently
inverted to get a clear suspension.
5. To the suspension, 350 µL of Solution III (3M Guanidine HCl (Sigma Aldrich Chemical Company,
USA) prepared in water and stored at room temperature.) was added and the contents of the
tube were gently mixed by inverting for a few seconds.
6. The tube then was centrifuged for 5-10 min at 12,000xg and the clear supernatant was
transferred to a fresh centrifuge tube.
7. To the reaction mix, equal volumes of absolute alcohol was added and incubated at room
temperature for 10 min.
8. The DNA pellet from the reaction mixture was collected by centrifugation (10 min at 12000xg)
and the supernatant was removed by pipetting.
9. The pellet was washed with chilled ethanol (70%, v/v) by centrifuging at 12,000xg for 5 min.
10. The DNA pellet was dried at 650C for 3 to 5 min or air dried until the ethanol evaporated.
11. The purified DNA was dissolved in 30-50µl of Tris or TE buffer

c. Apakah fungsi dari 3M Guanidine HCl pada langkah kerja no. 5?


What is the function of 3M Guanidine HCl at step work no.5?

d. Dinda ingin mengetahui apakah hasil isolasi plasmid tersebut membawa gen insert
yang telah disisipkan. Konfirmasi apakah yang akan dilakukan oleh dinda?
Dinda want to find out whether the plasmid extracts carried the insert gene that had
been inserted. What will dinda do to confirm it?

e. Informasi apakah yang dapat anda simpulkan berdasarkan table di atas?


What the information you can conclude based on the table above?
3. Untuk memetakan plasmid, anda melakukan serangkaian reaksi restriksi dengan hasil
seperti pada table di bawah ini. Perkirakanlah Panjang dan gambarkan peta restriksinya!

For plasmid mapping, you perform a series of restriction reactions with results as shown
in the table below. Estimate Length and draw the restriction map!

a. Jika hasil yang didapatkan adalah sebagai berikut!


If the results obtained are shown!

ScaI EcoRI BamHI KpnI EcoRI EcoRI BamHI EcoRI ScaI ScaI ScaI ScaI ScaI ScaI ScaI
BamHI KpnI KpnI BamHI EcoRI BamHI KpnI BamHI EcoRI EcoRI EcoRI
KpnI KpnI BamHI KpnI KpnI
BamHI
5.6 5.6 5.6 5.6 2 5 3 2 0.3 2.3 5.3 2.3 0.3 0.3 0.3
3.6 0.6 2.6 3 5.3 3.3 0.3 3 2 5 2
0.6 0.3 3.3 0.3 3
0.3

b. Jika hasil yang didapatkan adalah sebagai berikut!


If the results obtained are shown!
NdeI NcoI EcoRV NdeI + NcoI NdeI + NcoI + EcoRV NdeI + NcoI +EcoRV
EcoRV
5.8 0.8 7.8 2 0.3 2.3 2.7
2 2.3 2.7 1.7 0.8 0.8
4.7 3.1 5.8 3 2.3
1.7 1.7
0.3

c. Jika hasil yang didapatkan adalah sebagai berikut!


If the results obtained are shown!
PstI SalI HindIII PstI + SalI PstI + HindIII SalI + HindIII PstI + SalI + HindIII
1.4 2.8 0.5 0.2 0.1 0.3 0.2
3.4 2 1.6 1.4 1.3 2.5 0.1
2.7 1.2 1.4 0.2 1.3
2.0 0.5 1.3 1.2
1.5 0.5 0.2
0.5
1.3
4.a Rara telah selesai melakukan isolasi RNA dari daun buah anggur. Selanjutnya ia melakukan
kuantifikasi dan pengecekan kualitas RNA, Ia melakukan elektroforesis. Gambar dibawah
merupakan elektroforegram isolat RNA daun buah anggur. Menurut kalian apa yang terjadi
pada hasil elektroforegram tersebut? Berikan masing-masing 2 (dua) alasan dan solusi untuk
Rara! (Catatan : Dilarang menyebutkan alasan akibat human error)

Rara has been finished doing RNA isolation from grapevine leaves. Then she did
quantification and checking the quality of RNA, she did electrophoresis. This picture below
show you the electrophoregram of RNA isolation from grapevine leaves. According to you,
what happened to the result of this electrophoregram? Give each 2 (two) reasons and solutins
to Rara! (Note : Please, do not mention the reason caused by human error)

Well Problem Solution


1. 1.

3
2. 2.

1. 1.

4
2. 2.

1. 1.

5
2. 2.

1. 1.
8
2. 2.

b. Berikut merupakan hasil spektrofotometer dari isolat RNA dari berbagai macam buah, baik
angiospermae maupun gymnospermae. Berdasarkan pengetahuan Anda, jawab pertanyaan
dibawah ini:
a. Jenis isolat buah yang menghasilkan hasil elektoferogram yang tipis?
b. Jenis isolat buah yang menghasilkan hasil elektroferogram yang tebal?
c. Jenis isolat buah yang terkontaminasi dengan protein?
d. Jenis isolat buah yang terkontaminasi dengan senyawa berat seperti metabolit
sekunder?

This table below show you the RNA quality and purity from difference fruits, which
angiospermae or gymnospermae. Based on your knowledge, answer the question below:
a. Which fruits will produce low intensity RNA?
b. Which fruits will produce high intensity RNA?
c. Which fruits will be contaminated by protein?
d. Which fruits will be contaminated by secondary metabolite?

Fruits Yield (µg) A260 A280 A230


Apple 85 0,032 0,02 0,027
Cherry 123 0,05 0,25 0,25
Pine 21 0,036 0,014 0,024
Spruce 12 0,042 0,21 0,21
Horse 53 0,024 0,018 0,016
chestnut

5. a. Jelaskan dan gambarkan perbedaan dari 3 jenis primer yang digunakan untuk sintesis
cdna!
Explain and illustrate the differences between 3 (three) kinds of primer that used for
cDNA synthesis!

b. Diantara 3 jenis primer yang sudah kalian sebutkan. Manakah primer yang dapat
digunakan untuk organisme prokariot dan organisme eukariot? Sertakan alasan
pemilihan primer tersebut!
Among 3 (three) kind of primers you mentioned before, which primer that can be used
for prokaryote organisms and eukaryote organisms? Mention it with the rationalization.
c. Jelaskan perbedaan, kelebihan, serta kekurangan dari metode antara ‘One step dan
two step RT-PCR’ !
Explain the differences, advantages, and disadvantages from ‘One step and two step RT-
PCR’ for cDNA synthesis!

d. Dibawah ini merupakan daftar trobleshooting dari hasil sintesis cDNA . Lengkapi
kolom dibawah ini Trouble shooting hasil isolasi cDNA!
This table below show you the list of trouubleshooting of cDNA synthesis. Complete the
troubleshooting of cDNA synthesis result in the column below!
Problem Possible Cause Recommendation
No cDNA Synthesis 1. 1.

2. 2.

3. 3.

Poor Specificity in PCR 1. 1.

2. 2.

3. 3.

Product in no-RTase 1. 1.
control
5. Sekelompok peneliti melakukan upaya dalam mengekspresikan protein heterolog pectin
lyase (pnl) pada sel Escherichia coli. Pada gambar (A) menunjukkan construct plasmid
yang digunakan dalam eksperimen sementara gambar (B) menunjukkan analisis total
protein dari E. coli transforman.

A group of researcher make an effort to express heterologous protein pectin lyase (pnl) on
Escherichia coli. Plasmid construct for the experiment can be seen in picture (A) and total
protein analysis from E. coli transformant can be seen on picture (B).

Legends :
Lane 1 : Protein Marker
Lane 2 : Before IPTG induction
Lane 3 : 1 hour post-induction
Lane 4 : 2 hour post-induction
Lane 5 : 3 hour post-induction

Picture A. Plasmid construct used in the experiment Picture B. Plasmid construct used in the experiment

(a) Apa pentingnya keberadaan sikuens signal yang terdapat downstream dari gen pnl dalam
proses rekayasa genetika ekspresi protein heterolog ?
What is the importance of signal sequence which located downstreams of pnl genes in
genetic engineering of heterologues protein expression ?

(b) Paparkan langkah – langkah yang dilakukan dalam eksperimen tersebut dari awal hingga
memperoleh hasil analisis total protein dengan menggunakan diagram alir !
**di awal, gen pnl dan plasmid pET29C masih dalam keadaan terpisah !

Describe each steps carried out in the experiment to obtain the results of the analysis of
the total protein using a flow chart !
**at the beginning, the pnl gene and plasmid pET29C are still in separate conditions)!
(c) (i) Berdasarkan gambar A dan B, berapa kira - kira ukuran gen pnl (bp) dan protein pectin
lyase (kDA)?
(i) Based on picture A and B, what is the approximate size of pnl gene (bp)and pectin lyase
protein (kDA) ?

(ii) Jelaskan apakah terjadi modifikasi pasca translasi yang mengubah ukuran protein pada
sel transforman ? Kaitkan juga dengan ukuran nukleotida dan protein untuk menyokong
jawaban anda !
*1 asam amino memiliki ukuran ± 110 Dalton, bervariasi berdasarkan rantai R asam
aminonya

(ii) Is there any post-translational modifications which change the size of protein on cell
transformants ? Give your explanation and relate it with nucleotide and protein size to
support your answer !
*1 amino acids size is approximately ± 110 Dalton, which is varied based on its R chain
of amino acids

(d) Peneliti melakukan immunostaining protein total dengan menggunakan anti six his-A dan
anti-PNL. Apa yang ingin ditunjukkan peneliti dengan membandingkan hasil
immunostaining anti six His-A dengan anti-PNL ?

The researcher conducted immunostaing for total protein using anti six his-A and anti-
PNL. What do you think the researcher want to shows by comparing the results of anti-six-
His-A and anti-PNL immunostaining ?

Picture A. Immunostaining results with Anti-six-His-A Picture B. Immunostaining results with Anti-PNL
Pemahaman Jurnal dan Presentasi.

Berikan jawaban yang lengkap untuk dua dari empat soal berikut:

a. Jelaskan metode Golden Gate dan gambarkan satu contoh mekanismenya.


b. Jelaskan metode Site-specific recombination dan gambarkan satu contoh mekanismenya.
c. Jelaskan hubungan antara laju inisiasi, laju elongasi, densitas ribosom dan laju sintesis protein
pada gen endogenus serta dikaitkan dengan adaptasi kodon.
d. Sebutkan 4 kelebihan Escherichia coli untuk memproduksi protein rekombinan.

Understanding of Journals and Presentations.


Give a complete answer to two of the following four questions:

a. Explain the Golden Gate method and draw an example of the mechanism.
b. Describe the Site-specific recombination method and draw an example of the mechanism.
c. Explain the relationship between initiation rate, elongation rate, ribosome density and rate of
protein synthesis in endogenous genes, and associate them with codon adaptation.
d. Mention 4 advantages of Escherichia coli for producing recombinant proteins.