Detailed Report on Cloning Expression of Fermentation Gene of Interest

This report elucidates the process of cloning expression, focusing on the technique of using an expression vector to clone and express a specific protein, particularly a fermentation gene of interest. The report highlights blue-white screening as an effective method, enabled by alpha complementation, for achieving protein expression. It details the insertion of the gene, emphasizing the role of the lac Z operon and plasmid vectors like pBR322. The transformation process, using methods such as CaCl2 or electro-competent cells, is explained, followed by a description of screening techniques involving chromogenic substrates like X-gal and IPTG. The report further explains the use of antibiotic-resistant genes for understanding transformation procedures. Finally, the process of confirming fermentation gene expression through colony PCR and plasmid miniprep is discussed, noting potential false positives and the importance of verifying the fermentation ability of transformed E. coli.

Running head: CLONING EXPRESSION
Cloning expression
Name of the student:
Name of the university
Author note:

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CLONING EXPRESSION
Process of expression of protein:
Expression cloning is defined as a technique in the cloning through use of expression
vector for generating a clone and expression of one protein (McGann et al., 2016). According to
Pyne et al. (2015), blue-white screening is the most effective technique for obtaining the
expression of a protein which works through alpha complementation. This paper will illustrate a
process of cloning expression of a fermentation gene of interest using a plasmid in the following
paragraphs:
Insertion of the gene:
The presence of the lactose in the surroundings triggers the lac Z operon in the E.coli.
Most of the plasmid vectors have a short segment of lac Z gene which contains the short
expression of lac Z which contains the coding information of B galactosides and E.coli strains
are used since it contains the lac Z deletion (Gaida et al., 2015).
For cloning, the pBR322 plasmid can be used (Li et al., 2015). After cutting the plasmid
with a restriction enzyme, the foreign gene can be in the multiple cloning sites of Lac Z and the
gene can be inserted within lac Z or outside which can be evaluated through screening. If gene of
interest is inserted into the vector or other than the location of MCS, the lac Z gene in the
plasmid vectors will show the deletion mutation in the host E.coli and will produce a functional
enzyme of beta-galactosidase (Li et al., 2015). An antibiotic-resistant gene (Amp r) is inserted
after ligation; it can be ligated through T4 ligase.

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CLONING EXPRESSION
Figure: gene insertion
Source: (Gaida et al., 2015).
Transformation and screening:
The transformation procedure can be conducted through either cacl2 or electro
competent cell methods. After transformation procedure, for screening, the clones containing
gene of interest, a chromagenic substrate such as X-gal along with isopropyl-b-D-1
thiogalactopyranosides (IPTG) used for the blue-white screening in the plate along with the
antibiotic. IPTG is a non-metabolized analogue of the galactose which assists in inducing the
expression of Lacz. The ampiciline genes were used for understanding the transformation
procedures (Guntas et al., 2015). Only transformed cells would grow in the agar plate and non
transformed cells would not be appeared in the plate. For visualization, chromogenic substrate
x-gal can be used (McGann et al., 2016). The screening can be done by pour plate method where
both white and blue colonies can be found. Blue white screening method worked by alpha
complementation process whereas if the gene was inserted in the lac Z, it will disrupt the
production of beta- galactosidase (Gaida et al., 2015). White colonize give the indication of the

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vector containing fermentation gene. ON the other hand, x gal is cleaned by beta- galactosidase
because of alpha complementation and gives a blue colour (Zhou et al., 2017). Therefore, blue
colonizes appeared in the plate contain uninterrupted Lac Z with no insertion.
Figure: screening
Source: (Gaida et al., 2015).
Further process:
For confirming the fermentation gene expression, colony PCR can be done. Sometimes,
colony PCR can give a false positive result and therefore, plasmid miniprep and then PCR can be
done and the protein expression can be observed through evaluating that the non fermenting
e.coli now have the ability to ferment .

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CLONING EXPRESSION
References:
Gaida, S.M., Sandoval, N.R., Nicolaou, S.A., Chen, Y., Venkataramanan, K.P. and Papoutsakis,
E.T., (2015). Expression of heterologous sigma factors enables functional screening of
metagenomic and heterologous genomic libraries. Nature communications, 6, p.7045.
Guntas, G., Hallett, R.A., Zimmerman, S.P., Williams, T., Yumerefendi, H., Bear, J.E. and
Kuhlman, B., (2015). Engineering an improved light-induced dimer (iLID) for
controlling the localization and activity of signaling proteins. Proceedings of the National
Academy of Sciences, 112(1), pp.112-117.
Li, Y., Lin, Z., Huang, C., Zhang, Y., Wang, Z., Tang, Y.J., Chen, T. and Zhao, X., 2015.
Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome
editing. Metabolic engineering, 31, pp.13-21.
McGann, P., Snesrud, E., Maybank, R., Corey, B., Ong, A.C., Clifford, R., Hinkle, M.,
Whitman, T., Lesho, E. and Schaecher, K.E.,( 2016). Escherichia coli harboring mcr-1
and blaCTX-M on a novel IncF plasmid: first report of mcr-1 in the United
States. Antimicrobial agents and chemotherapy, 60(7), pp.4420-4421.
Pyne, M.E., Moo-Young, M., Chung, D.A. and Chou, C.P., (2015). Coupling the CRISPR/Cas9
system to lambda Red recombineering enables simplified chromosomal gene replacement
in Escherichia coli. Applied and environmental microbiology, pp.AEM-01248.
Zhou, C., Pu, W., Peng, C., Li, W., Xiao, L., Xu, H., Sirois, P., Xing, C., Lu, G., He, N. and
Liao, D., (2017). A Sensitive Assay Enriched with Blue/White Screening to Empower
Sequencing Analysis for G> A Hotspot Mutation in Codon 13 of KRAS Gene. Journal of
Nanoscience and Nanotechnology, 17(12), pp.9176-9181.