Volume 6, Issue 3, March – 2021 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Application of Recombinant DNA Technologies on

Sub-cloning of Transcriptional Co-factor
Michael Halim1
1
University of Salford, MSc Biomedical Science, Greater Manchester, United Kingdom
Corresponding author: Michael Halim

Abstract:- The expression or repression of a wide variety of necessary the transcriptional activity also has slight affection
genes, which code for protectins, controls vital physiological because of GR inhibition (Sanchez-Garcia et al., 2016).
processes like metabolism, development and immune responses. When hormone are not present then the binding of receptor
The transcription of genes started by activation of the is done by the coprocessor of GR that is NCoR (nuclear
glucocorticoid in both negative and positive manners like receptor corepressor) and SMRT (silencing mediator of
metabolism, immunization, and inflammation. The retinoid and thyroid hormone receptor). It also repress GR
transcription process is complex and involves a large number of dependent transcription by employing histone deacetylates
factors interconnected by a large number of co-factor factors. In to transcriptional complex NCoR and SMRT have CoRNR
response to stress TTC5 is an active factor because interaction boxes or LXXI/HIXXXI/L helix motifs basically they are
and stabilization and regulation of transcriptional activity of GR nuclear receptor that interacts domains. Helix motif form
depends on this co factor. There are many other factors are also different helices which binds nuclear receptors in
present but TTC5 is the main co factor because regulation of hydrophobic pocket along with C-terminal activating
GR in gene depends on it. The TTC5 control of GR is expected function domains. A major change occurs in this domain as
to contribute to glucose corticus and GR's physiological the attachment of coprocessor are inhibited by ligand
function. The TTC5 regulates the transcription of GR target binding it also controls release of nuclear receptor for
genes, including inflammation, involved in various processes. cytoplasmic complex. The interaction of co factor with GR
Duo TC5 has novel potential targets for different compounds is possible with AF1 or AF 2 domains. If it binds with AF2
which enable better control over glucocorticoid-related domain then it is called hormone dependent the members of
therapies, considering the importance of glucocorticoids in p160 family which are actually coactivators, p300/cAMP
treating inflammatory disorders and in general clinical practice. they activate against element binding protein BRG1
Wild type (2kb) is present on the agarose gel (but it is very (Brahma-related gene 1), PCAF (p300 CBP associated
blurry). Mutant type (2kb) is present on the agarose gel. factor), and vitamin receptor interacting protein 205
PET28a (5kb) is present on the agarose gel. (DRIP205)/TRAP220 (thyroid receptor associated protein
220), interact with this domain. A surface is generated by
I. INTRODUCTION hormones that has an ability of binding with LLXLL motif
they are present in coactivators after this process changes
Glucocorticoid receptor belongs to superfamily occurs in GR. The interaction between NR and coactivators
receptor that is nuclear hormone .basically glucocorticoid are possible by LXXLL motifs. On N terminus of receptor
are hormones known as lipophilic hormone adrenal cortex AF1 independent activation hormone is present. But it’s not
are the main component that releases this receptor at the restricted to the structure among NR family. The main factor
time of stress inside cytoplasm adrenal cortex are attached for the binding of AF1 has not been fully recognized yet.
with glucocorticoid in the cytoplasm . The transcription of But on the other hand members of P160 family like
genes started by activation of the glucocorticoid in both PRIP150 and tumor gene 101 are recognize as interacting
negative and positive manners like metabolism, domain (Halimah, Rahmat and Redjeki, 2019). The linked
immunization, and inflammation. In different steps like between the two domains is possible because the members
stability of protein, cofactor interaction, translational of p160 family and DRIP/TRAP complex both can interact
modification they are highly active because of different with AF-1 and AF-2 domains. The 160 family is well known
range of glucocorticoid action. During the transcriptional group which includes steroid receptor coactivator 1 (SRC-
activity the stability of glucocorticoid is really very 1), SRC-2, and SRC-3, which bind AF-2 of GR in a ligand-
important. When the exposure of GR with hormone is very dependent manner. Proteins uses intrinsic histone acetyl
long then the regulation of GR is low during proteasome transferase activity for co activation of GR. the GR can
dependent process. Protein stability has great impact at the directly bind by the employment of other cofactors that are
time of phosphorylation of receptor at the time of HATs p300/CBP (18). P300/CBP it can also act as co
proteosomal degradation of GR the key role is played by activators by acetylene histones and enrolling other HATs
ubiquitin ligases that are murine double minute w and RNA polymerase 2 to initiation point of transcription.
(Mdm2),CHIP (cterminus of hsp 70-interacting protein) and For numerous transcription factor p300 is really very
E6-AP. Other than regulation of GR proteasome machinery important p53 also includes in this family it contains
is responsible for of transcriptional activity of GR as well. transcriptional factor and serves as a component of multi
For the rearrangement of GR at promoter the proteasome is protein factor PCAF, tetratricopeptide repeat domain 5

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Volume 6, Issue 3, March – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
(TTC5), and junction mediating and regulatory protein by restrictive enzymes to incorporate the desired DNA
(JMY) are includes in this factor. After phosphorylation by (Puetz and Wurm, 2019).
ataxia telangiectasia DNA is damage as a result TTC5 is
stabilized. Composition of TTC5 is depend on six protein- PHATTC5 was developed as defined and permits
protein interaction. In Yeast cell division motifs were first temporary overexpriming of TTC5 in mammalian cells
recognized and promotes anaphase complex. Motif present (Demonacos et al 2001). We cut TTC5's CDNA from this
in tandem array are made up of 34 amino acids that have plasmid using the restrictive enzymes BamH1 and Xho1 and
similarities in size, arrangement and stability (Qi et al., put it into the same-enzyme PET 28A plasmid cut, which
2015). TPR motif are involves in many functions like enables bacteria to be exposed and the overproduction of
transportation of protein. Folding of protein, degradation TTC5 is easily purified and studied, for TTC5 to become
and transcriptional regulation. Many other motif proteins are overproduced. Remember that in-frame cloning without
also involved in regulation of GR. PCR, (classical cloning technique), is required in this
strategy. If there is no feasible restriction areas, then the
II. MATERIALS AND METHODS PCR-based approach should be used (Rasala and Mayfield,
2015).
The expression or repression of a wide variety of
genes, which code for protectins, controls vital physiological Aim
processes like metabolism, development and immune The aim of this experiment was to subclone p53 and
responses. The gene transcription system, which is regulated GR transcriptional cofactor TTC5 from mammalian
by transcription factors in effect, controls various cellular expression vector into the bacterial expression vector to
processes and environmental stimulus responses. The facilitate future studies of TTC5 function
transcription process is complex and involves a large
number of factors interconnected by a large number of co- Objectives
factor factors (Chao, Yuan and Zhao, 2015). For eg, tumor  Design strategy to make recombinant DNA
suppressor p53 and glucocorticoid receptor are sequence  Perform restriction digestion of the DNA
defined transcription factors. To order to function  Use electrophoretic techniques to separate different DNA
efficiently, these variables must communicate fragments
withTTC5/STRAP co-factor, which can be evaluated in this  Isolate DNA fragments from the gel
practical manner. Demonacos et al. in 2001 described TTC5  Learn how to ligate two different pieces of DNA
as the p300 pressure sensitive activator (Demonacos, 2001;  Introduce plasmid DNA into bacteria
Calderwood, 2013) as motif 5 for tetratricopeptides. The
protein of 440 amino acids was found to consist entirely of Steps
six TPR factors implicated in the development of Step 1: Set up restriction digest to transfer TTC5 cDNA
multiprotein complexes and cancer (Wen, 2018). from mammalian expression vector (HAPCDNA3) to
PET28a vector so that TTC5 can be expressed in bacteria
Plasmids Step 2: Prepared agarose gel (this will be prepared for you)
Double-stranding circular DNA molecules found in Step 3: Run the gel, cut out and isolate fragment
bacterial cells are plasmids or vectors. These can be Step 4: Ligate DNA overnight (you will prepare ligation
replicating and are used in molecular biology with multiple mix, but one will be prepared for you previous day)
copies. They have a replication origin (ori) which allows Step 5: Introduced DNA into bacteria (known as
them to replicate and produce large amounts of DNA in transformation of bacteria)
bacteria (World Health Organization, 2015). We have
antibiotic markers that can live on plates that involve this Step 1: Set up a restriction enzyme digest
antibiotic in bacteria used in selection process only. Label 3x1.5 ml eppendorf tubes (very important in order not
Furthermore, plasmids contain multiple cloning sites (MCS) to mix up your samples). Pipette DNA, buffer, water and at
which are DNA sequence clusters, which can be decreased the end add enzyme using p20 pipette according to the
scheme below.

HAPCDNA3TTC5wt(tube1) HAPCDNA3TTC5 A243G mutant (tube 2) Pet28a vector (tube 3)

DNA 14µl 14µl 14µl
Buffer 2µl 2µl 2µl
BamH1 1µl 1µl 1µl
Xho1 1µl 1µl 1µl
Water 2µl 2µl 2µl
Total 20µl 20µl 20µl

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Volume 6, Issue 3, March – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Mix the liquid and then spin briefly in the
microcentrifuge. Incubate samples for 1 hr in a 370C water Step 5: Introduce DNA into bacteria (known as
bath transformation of bacteria)
Using sterile tubes containing 50 µl DH5α E.coli from
Step 2: Prepare agarose gel (this will be done by technical Thermo Fisher Scientific chemically competent host cells.
staff for you) Key:
Agarose gel is prepared by technical staff for students W/type = 10a
M/type =10b
Step 3: Run the gel and isolate fragment pHSG298 = 10c
1. Removed the tubes from the water bath, add 5 µl of pET28a = 10d
loading buffer, mix and pulse spin
2. Removed the comb, placed the gel in electrophoresis Before plating out, labelled each LB plate
tank filled with the 1x TAE, with the wells next to appropriately with name.
negative electrode.
3. In the first lane load 10 µl of the Hyperladder 1 (DNA Thaw competent bacteria cells on ice (50 µl per
size marker) and in the rest of lanes load the entire ligation mix)
samples into respective wells. 1. Add 5 µl of the ligation mix to 50 µl of competent
4. Placed the lid on top of the tank, switched the power bacteria mix by flicking.
pack and set it to 100V. Checked if the current is 2. Place cells on ice for 1 min
flowing. After 20min stopped the electrophoresis (make 3. Heat shock in a water bath at 420C for 40 seconds
sure blue dye does not run of. 4. Place back on ice for 1 min
5. Took the gel out and placed it in the UV transiluminator 5. Add 500 µl of LB
to visualise the DNA. 6. Place on horizontal shaker for 30 min at 370C
6. Noted that only linear DNA will run in line with the 7. Plate 250 µl of each mix onto LB-kan plates
marker and therefore uncut circular plasmid size cannot 8. Allow 5 min for absorption then place plates upside
be estimated down in a 37oC incubator for 24 hrs
7. Cut out the bands: lane 2 and 3 (HAPCDNA3TTC5) 9. Next day pictures will be taken of a plate with -this will
around 2kb-this is your insert; cut out the PET28a be figure 2 in your report
plasmid band (around 5kb)-this is the plasmid you are
inserting the insert to. III. RESULTS
8. Isolate the DNA using QIAquick gel extraction kit (or if
no time purified fragments will be provided for you) Results are obtained after step 3 and step 5 respectively.
9. Weigh the excised fragment and add 3 volumes of QG
buffer Results after Step 3
10. Vortex every 2 min and incubate at 500C for 10 min to
dissolve the gel
11. Add one volume of isopropanol and mix
12. Add this to the column that is in its 2 ml tube, and spin
1min 13000 rpm
13. Discard flow through add 750 µl of PE buffer to wash
the column, spin again 1min 13000 rpm
14. Discard flow through and place the column in the clean
1.5 ml tube
15. Elute the DNA by adding 50 µl of the EB buffer and
spinning again 1min 13000 RPM
16. Use eluted DNA for ligation

Step 4: Ligate DNA

1. Label three 1.5 ml tubes and pipette into all of them 1 µl
of 10X ligation buffer, then 4 µl of distilled water into Agarose gel electrophoresis of wild type, mutant type and
the first tube and 2 µl of distilled water into tubes 2 and 3 PET28a
2. Pipette 2 µl of the cut PET28A vector into tube number 1
3. Pipette 2 µl of the cut PET28A vector and 2 µl of the Column 1: Hyperladder 1 (DNA size marker)
insert 1 (WT) into tube number 2 Column 2: Wild type
4. Pipette 2 µl of the cut PET28A vector and 2 µl of the Column 3: Mutant type
insert 1 (mut) into tube number 3 Column 4: PET28a
5. Add 1 µl of the enzyme ligase into all tubes, mix pulse
spin and incubate on the room temperature for few hours. Results interpretation: Wild type (2kb) is present on the
agarose gel (but it is very blurry). Mutant type (2kb) is
present on the agarose gel. PET28a (5kb) is present on the
agarose gel.

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It is provided that the DNA is extracted from organism Lane 4 Pet only
as per the study which is being cut into small fragments of a If there are 2 bands it is possible the plasmid was not
size that will be suitable for the procedure of cloning. In cut-perhaps enzymes were not added due to pipetting error
most of the cases the cleaving the DNA is being performed or enzymes were inactivated by being left at room
to achieve this with a restriction enzyme (Jia and Jeon, temperature too long – uncut plasmid can be supercoiled
2016). Whereas different types of strains and species of DNA that can give rise to several bands o a gel
bacteria are used to extract restriction enzymes, in which
they act as defense mechanisms against viruses. Plates
No colonies can be seen if:
Results after Step 5  wrong plates-wrong antibiotic-but unlikely as there are
colonies in other plates
 ligation not done properly, but positive ctrl should have
colonies transformation not done properly-pipetting
error, or competent cells left on room temperature too
long, of spreading not done properly etc.

IV. DISCUSSION

In response to stress TTC5 is an active factor because

interaction and stabilization and regulation of transcriptional
activity of GR depends on this co factor. There are many
other factors are also present but TTC5 is the main co factor
because regulation of GR in gene depends on it (Kurachi
and Kinoshita, 2019).
10A – wild type It was observed that in GR and ER ttc5 is hormone
10B – Mutant type inducible factor where as in AR it non hormone inducible
10C – PHSG298 (positive control): pUC-type bacterial factor. In GR interaction different motifs are included for
cloning vector with kanamycin resistance gene, the binding of GR the LXLLIS the most important factor
10D – cut pET28a plasmid (negative control) because modern mutation occurred in this arrangement
which can be altered later with the receptor (Withers III et
It is analyzed that staggered cuts are caused by most al., 2017). On the other hand many other motifs are also
useful restriction enzymes as they leave a single-stranded present like TRP motifs 2,3 and most important is 6 because
overhang at the site of cleavage. If the donor DNA as well most important mutation minimize in this motif. Whether
as the vector DNA are both cut with the similar enzyme. In hormone are present or not bing of GR is possible in both
addition to this it is also proven that glucocorticoid receptor possibilities buy the most activity and affinity was observed
are related to superfamily receptor which originally is a in the presence of hormone. TTC5 might be necessary as
nuclear hormone (Stehr, 2015). Basically glucocorticoid are molecular scaffold protein and can employ other cofactor
hormones which are called lipophilic hormone adrenal during transcriptional process along with numerous
cortex which are the key component releasing this receptor enzymatic activity. In protein - protein interaction TPR
in stressing conditions inside cytoplasm (Yun and Yoon, motif plays the key role and total six motif are recognized in
2017). this structure. Hsps and p300 are also associated with TTC5
and GR the most difficult structure can be easier by the
The results mentioned that wild type (2kb) is present involvement of numerous motifs in GR and TTCF
on the agarose gel. While on the other hand mutant type interaction many other substance can employ in context
(2kb) is present on the agarose gel. Moreover, it is also dependent manner (Kaddour et al., 2019). The most
identified in this report that when 2 bands are present there important factor for regulating the stability of P53 is TTCF
are chances of the fact that there is no cut on plasmid. same act is performed in GR regulation. The stability of GR
is increased by TTC5 and important role is played in
Potential explanations for different results stabilization of endogenous GR. For stabilization impact
interaction of TTC5 and GR is really very important.
Gel Because their no interaction between the TTCF mutant 1380
Lane 1 Hyperladder is used to determine molecular but able to stabilize GR when hormone is present (Marshall
weight, note depending how long the gel is run the patter et al., 2018). Our observation indicates that TTC5 protects
between provided example and your data may differ the receptor from the action of ubiquitin ligases like MDm2,
which produce in minimization proteasome mediated
Lane 2/3 no bands or smear-cause: degradation of GR. Stability of NRs and transcriptional
1. Didn’t cut the DNA factor are associated with transcriptional factor (Timm and
2. Pipetting error Niemeyer, 2015). Whenever transcription is inhibited
3. Not high enough concentration of DNA to start with or ubiquitination of Est-bound is delayed for cycling of
degraded DNA promoter degradation of ubquinated ER is very important

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Volume 6, Issue 3, March – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
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