plasmid

QUESTION 1:
A) Choose a protein of interest (one that has a therapeutic, diagnostic, industrial or
agricultural use).
B) Describe the use of the protein
C) Choose any commercially available plasmid.
D) Justify the choice based on the type of protein you wish to express and purify.
ANSWER 1:
A) Regulatory proteins
B) Regulatory proteins are mostly transcription factors. TATA binding protein is
important in transcription regulation of multiple proteins, and is located in most cell
types. Tip repressor protein is more specified, residing in the tip operon in E. Colt
This provides negative feedback for the transcription of proteins responsible for
making tryptophan (Protein Atlas, 2015).
C) Commercially available Plasmid name: pBR322, Company name: Thermo
Scientific, List of Restriction enzyme: Eco R1, Hind –III, Bam H1
pBR322 Plasmid Map:
D) The SeqA protein was distinguished as a factor that counteracts re – start of recently
recreated, hemimethylated birthplaces. SeqA likewise appears to repress start of
completely methylated causes, accordingly adding to the control of chromosomal
replication. The SeqA protein was found to tie to two locales in the left piece of the
cause, close to the AT-rich area where strand division happens amid start of
replication. Similar restricting locales appeared to be favoured regardless of whether
the beginning was in the recently reproduced (hemimethylated) state or not.
Notwithstanding restricting particularly to gatherings of GATC locales, the SeqA
protein was fit for interfacing non-particularly with contrarily supercoiled DNA,
limiting the supercoils in a form like that seen with histone-like protein HU. The
limitation of supercoils by SeqA was, rather than that of HU, helpful (Lurz, Torheim,
Wold, Messer, & Skarstad, 2011).
QUESTION 2:
Summarise the function of each of the components shown on the plasmid you have chosen
(read the product literature for the plasmids this can be found on the web). You will need to
read any relevant scientific papers you can find and the recommended text book. (Half a page
max)
ANSWER 2:
pBR322 is a normally utilized cloning vector in E.coli. It is vital in light of the fact that, it
contains 4.36kb base sets and qualities for ampicilin and antibiotic medication protection and
opened up utilizing chloramphenicol. It is broadly utilized cloning vector.
We can screen the bacterium that contained the pBR322 plasmid with a bit of remote DNA
embedded into the EcoRV site by blue white screening strategy. Fruitful articulation of β-
galactosidase is an element of how in place its quality stays in the wake of cloning, an in-
place quality offers ascend to a blue province, an interfered with quality (i.e. corrupted DNA
end) offers ascend to a white province (New England Bio Labs, 2017).
Features of plasmid:
1. ampR sequence → permits selection of bacteria containing the plasmid
2. ori sequence → origin of replication
3. tetR sequence → permits selection of bacteria containing the plasmid
4. BamHI sequence → insertion of foreign DNA here permits identification of bacteria
containing recombinant plasmids (with insert).
5. PstI sequence → insertion of foreign DNA here permits identification of bacteria
containing recombinant plasmids (with insert).
6. PvuII sequence → insertion of foreign DNA here permits identification of bacteria
containing recombinant plasmids (with insert).
QUESTION 3:
Decide on which components you will use to clone in the gene, express and purify your
protein. LIST these in a table. Also note the function of those components, in the table.
ANSWER 3:
The sequence of gene is downloaded from Uniprot database YDEH_ECOLI Diguanylate
cyclase YdeH OS=Escherichla coil (strain KU) Sequence:
https://www.chegg.com/homework-help/questions-and-answers/diagram-plasmid-pbr322-shown-match-feature-plasmid-pbr322-left-one-appropriate-description-q16836573?trackid=797742e2&strackid=232b7dea&ii=6
https://www.chegg.com/homework-help/questions-and-answers/diagram-plasmid-pbr322-shown-match-feature-plasmid-pbr322-left-one-appropriate-description-q16836573?trackid=797742e2&strackid=232b7dea&ii=6
MIKKTTEIDAILLNLNKAIDAHYQWLVSMFHSVVARDASKPEITDNHSYGLCQFGRW
IDH
LGPLDNDELPYVRLMDSAHQHMHNCGRELMLAIVENHWQDAHFDAFQEGLLSFTA
ALTDY
KIYLLTIRSNMDVLTGLPGRRVLDESFDHQLRNAEPLNLYLMLLDIDRFKLVNDTYG
HLI
GDVVLRTLATYLASWTRDYETVYRYGGEEFIIIVKAANDEEACRAGVRICQLVDNH
AITH
SEGHINITVTAGVSRAFPEEPLDVVIGRADRAMYEGKQTGRNRCMFIDEQNVINRV
A strategy to clone and express the gene is as follows:
 Designing of the construct is done by
 restriction analysis,
 domain analysis,
 PCR primer design and
 selection of the vector
Primers are designed for ydeH gene and their sequences are given as follow:
i. Primer 1: GCCATCAAGAAGACAACGGAAAT
ii. Primer 2: CCAACTCGGTTAATCACATTTTG
Cloning of gene by PCR:
 cycle number
 annealing temperature
 template concentration
 choice of Taq polymerase
 Analysis of the PCR on agarose gel using molecular weight marker.
Cloning of this amplified PCR product in a plasmid pUC19 as it is a high copy number
plasmid with selectable markers. The colonies are selected and screened by using Blue-White
Screening. White coloured colonies are selected as they are recombinant ones.
Protein expression:
 Transform appropriate DNA plasmid into E. coli cells. these cells must be competent.
 Make a starter culture to about 250 to 500 ml of LB broth for protein expression.
Antibiotics should be added.
 Make the big batch of bacterial culture for protein expression.
Protein purification:
 Lysis and sonication of the bacteria
 Affinity chromatography
 Purification can be performed under native or denatured conditions.
 SDS PAGE or Western Blot will be performed for the quality testing.
QUESTION 4:
Do a web search at the patent search web sites (discussed in lectures and/or workshops) and a
search of supplier catalogues to see if any of the following components of your plasmid are
already described in patent documents:
a) Promoter sequences in the plasmid (there might be more than one promoter in...