Samet Yilmaz

7262963
October 8th, 2015

Lab #2: Ligation assignment BCH3356/BPS3151

1. a) The PMID for the article is: 22426482
b) The PMID number for the sequencing article is: 3903658 and for
the cloning article is 3011596.
The first article is published in 1985 and the second one in 1986, the
sequencing article is free and directly available on pubmed however
the cloning article is accessible through an external link. Both articles
are accessible in full just at different locations.
c) The article discussing the mutation of T7 RNA polymerase into a T3
promoter has a PMID of 1453460, the mutation occurs at position 748
of the T7 polymerase and is a switch from the amino acid (asn) to
(asp) which switches promoter specificity.
d) An article was found published by Dr. Boddy and is referenced
below
Boddy CN. Bioinformatics tools for genome mining of polyketide and
non-ribosomal peptides. J Ind Microbiol Biotechnol. 2014;41(2):44350.
2. A )
G TGT ATG AAT TCX ATG AAC ACG ATT AACATC GCT AAG AAC GAC TTC
TCT

The first 6 nucleotides are present for optimal ECOR1 activity

The next six are the EcoR1 recognition sequence
The X represents a random nucleotide added to make the RNA
polymerase reading sequence in frame
The remaining nucleotides are the coding sequence of T7 RNA
polymerase
B)
Figure 10. from the lab manual is used

MAT tag AAG CTT CTC GAG AAT TCA TG AAC -RNA pol sequence
The EcoR1 recognition site is highlighted in blue. Once this sequence
is cut out we are left with
MAT tag AAG CTT CTC GAA TGA AC -RNA pol sequence
The start codon is not in frame as it stands and coding cannot begin.
MAT tag AAG CTT CTC GAX ATG AAC -RNA pol sequence
The addition of a random nucleotide (A, T, G or C) puts the ATG start
codon in frame by forming a triplet.
The addition of this nucleotide allows the coding of the desired
protein to begin.
3. A) The only way for the reformation of the pre-digested plasmid vector
would be the ligation of the Hind III and EcoRI sticky ends which can
only be possible via a point mutation or tautomerization. The stick
ends of HindIII and EcoRI do not match up properly regularly and will
thus only ligate when the correct mutations occur.
EcoRI ends: GAG
AGCTT: HindIII
CTC TTA A
A
The G and T pairs will not bind as well as the C and T pairs however if
tautomerization is observed or a point mutation occurs we can
observe ligation and thus the survival of the vector. The mutations are
rather straight forward. If G becomes an A or the T mutates into C
there can be bonding between the two. If these two mismatched pairs
become matched through a mutation then the vector can circularize
again causing it to survive. The tautomer forms are also a possible
way for the nucleotides to mismatch. For example the imino form of
adenine can form the three hydrogen bonds necessary to bond with
cytosine. The enol form of guanine can also bind with thymine and
these tautomeric forms will allow the circularization of the plasmid
and thus its survival due to the presence of amplicin.
B)
88 colonies in 1 ng (10x dilution)
88 x 10 = 880 colonies / ng
880 colonies/ng x 1000 ng/g = 880000colonies/g
8.8 x 105 colonies/g