Problem untuk Mutagenesia

Berikut ini adalah cuplikan abstrak yang anda baca mengenai protein iso-1-sitokrom c.
--------------------------------------------------------------------------------------------------------Abstract
Yeast iso-1-cytochrome c is one of the least stable mitochondrial cytochromes c. We
have used a coordinated approach, combining the known functional and structural
properties of cytochromes c, to engineer mutations into yeast iso-1-cytochrome c with
the goal of selectively increasing the stability of the protein. The two redox forms of the
native protein and six different mutant forms of yeast iso-1-cytochrome c were analyzed
by differential scanning calorimetry (DSC). The relative stability, expressed as the
difference in the Gibb's free energy of denaturation at a given temperature between the
native and mutant forms (DeltaDeltaG(Tref)), was determined for each of the proteins.
In both oxidation states, the mutant proteins T102C, Q69E/T102C, K96A/T102C, and
Q69E/Q96A/T102C were more stable than the wild-type protein, respectively. The
increased stability of the mutant proteins is proposed to be due to the removal of a rare
surface cysteine and the stabilization of two distorted alpha-helices.
--------------------------------------------------------------------------------------------------------Anda, sebagai perekayasa gen, diminta oleh perusahaan anda untuk membuat salah satu
dari protein mutan tersebut.
Berikut ini informasi yang anda miliki mengenai sekuen protein dan asam nukleat dari
protein tersebut.
Sekuen Protein
1 MPAPYEKGSE KKGATLFKTR 21 CLQCHTVEKG GPHKVGPNLH
41 GVFGRKSGLA EGYSYTDANK 61 KKGVEWSEQT MSDYLENPKK
81 YIPGTKMAFG GLKKPKDRND 101 LVTYLKKATS
Sekuen Gen
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2.
3.

ttattcttat
ccatacgaaa
tgtcataccg
ggtagaaaat
gttgaatggt
ggtactaaaa
tatttaaaga
gggtggtatt

ttcttcaatt
aaggttcaga
ttgaaaaagg
ccggtttagc
ctgaacaaac
tggcttttgg
aagctacttc
tat

taatttaatt
aaagaaaggt
tggtccacat
tgaaggttat
catgagtgat
tggtttaaaa
ttaaatatca

taatataact
gctactttat
aaagttggtc
tcttatactg
tatttagaaa
aaaccaaagg
taataaaaca

atatatcaat
tcaaaactag
caaatttaca
atgctaataa
acccaaagaa
atagaaatga
agtgtttttt

aatgccagct
atgtttacaa
tggtgttttc
aaagaaagga
atatattcct
tttagttact
tgtttttttt

Buatlah platform urutan/diagram alir untuk mengerjakan tugas ini ?

Buatlah primer-primer yang diperlukan untuk membuat mutan protein tersebut ?
Sarankan dan jelaskan jenis sel yang digunakan oleh anda untuk mendapatkan Mutan Protein tersebut
diatas dan juga vektornya ?

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Usulkan juga bagaimana cara/metode untuk memastikan bahwa Mutasinya benar.

Bagaimana memastikan bahwa proses transformasi vektor yang telah dibuat itu berhasil.
atgccagctc
tgtttacaa
tgtcataccg
ggtagaaaat
gttgaatggt
ggtactaaaa
tatttaaaga
gggtggtatt

catacgaaaa aggttcagaa aagaaaggtg ctactttatt caaaactaga

ttgaaaaagg
ccggtttagc
ctgaacaaac
tggcttttgg
aagctacttc
tat

tggtccacat
tgaaggttat
catgagtgat
tggtttaaaa
ttaaatatca

aaagttggtc
tcttatactg
tatttagaaa
aaaccaaagg
taataaaaca

caaatttaca
atgctaataa
acccaaagaa
atagaaatga
agtgtttttt

tggtgttttc
aaagaaagga
atatattcct
tttagttact
tgtttttttt