Faculty of Science
PROTEIN TRANSPLANT: CLONING GENES FOR TWO NtaA PROTEINS FROM
GORDONIA SP. NB4-1Y
2015 | LAURA SMYLIE

B.Sc. Honours thesis

PROTEIN TRANSPLANT: CLONING GENES FOR TWO NtaA PROTEINS FROM
GORDONIA SP. NB4-1Y

by

Laura Smylie

A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENT OF BIOLOGICAL SCIENCES
(Cellular, Molecular and Microbial Biology)

This thesis has been accepted as conforming to the required standards by:
Jonathan Van Hamme (Ph.D.), Thesis Supervisor, Dept. Biological Sciences
Don Nelson (Ph.D.), Co-supervisor, Dept. Biological Sciences
Cynthia Ross Friedman (Ph.D.), Examining Committee member, Dept. Biological Sciences

Dated this 20th day of April, 2015, in Kamloops, British Columbia, Canada

i

ABSTRACT
Some telomerisation-based aqueous film-forming foams (AFFF) used to fight forest
fires contain 6:2 fluorotelomer sulfonate (6:2 FTS), or precursors that degrade to 6:2 FTS,
which has been found to accumulate in groundwater systems and appear in surface waters
and in the livers of fish following AFFF deployment.

By examining the microbial

metabolism of organosulfur compounds such as 6:2 FTS, therein lies potential for
development of bioremediation tools. To date, the metabolism of 6:2 FTS has been observed
in the soil bacterium Gordonia sp. NB4-1Y, an organism that was isolated in Kamloops,
British Columbia Canada. When NB4-1Y was supplied 6:2 FTS as the sole sulfur source, the
differential production of two nitrilotriacetate (NtaA) monooxygenases was observed when
compared to a MgSO4 supplemented culture. This led to the hypothesis that these enzymes
initiate the desulfurization reaction used by NB4-1Y for 6:2 FTS biodegradation.

To

examine this hypothesis, DNA was isolated from NB4-1Y using a modified Streptomyces
genomic DNA isolation protocol, and this DNA was used as a template to amplify the
protein-coding region for each NtaA enzyme by polymerase chain reaction (PCR). PCR
primers were designed to introduce restriction enzyme sites to the 5' and 3' ends of the genes
to allow for subsequent directional cloning. These genes were then transferred into DH5α
competent E. coli using a pBluescript cloning vector for sequencing.

Once the gene

sequence data has been obtained, the gene inserts will be moved into a pMAL-c2 expression
vector that will allow for the production and purification of large quantities of the NtaA
enzymes for characterization. The enzymes will be purified from an E. coli expression host
using affinity chromatography by taking advantage of a maltose binding protein introduced
to the N-terminal region of the target proteins using the pMAL-c2 expression vector, a
ii

binding protein that has an affinity for amylose. Once purified, spectrophotometric assays
monitoring the rate of NADH oxidation will be performed to definitively determine if these
enzymes are involved in 6:2 FTS desulfurization in Gordonia sp. NB4-1Y, helping us further
understand the potential value of Gordonia as a bioremediation tool.
Thesis Supervisor: Associate Professor Dr. Jonathan Van Hamme

iii

ACKNOWLEDGEMENTS
Thank you to my supervisor Dr. Jonathan Van Hamme (funded by NSERC) for this amazing
opportunity, and to my co-supervisor Dr. Don Nelson for all of his help and encouragement.
I would also like to thank my parents, Jim and Melissa, my two sisters, Katie and Emma and
my boyfriend Marian for their continued love and support.

iv

TABLE OF CONTENTS
ABSTRACT ............................................................................................................................ ii
ACKNOWLEDGEMENTS ...................................................................................................iv
LIST OF FIGURES .............................................................................................................. vii
LIST OF TABLES ............................................................................................................... viii
1

INTRODUCTION ............................................................................................................. 1

2

MATERIALS AND METHODS...................................................................................... 5
2.1 Bacterial Strains and Growth Conditions ................................................................. 5
2.2 DNA Extraction ........................................................................................................... 5
2.3 DNA Quantification .................................................................................................... 6
2.4 Characterizing plasmids ............................................................................................. 6
2.5 Polymerase Chain Reaction Amplification ............................................................... 7
2.5.1
2.5.2
2.5.3
2.5.4

PCR Primer Design .............................................................................................................7
PCR Reaction Setup ............................................................................................................9
Colony PCR Amplification of E. coli (DH5α) Using NB4-1Y Specific Primers .............10
ISGA 1222 & 1218 PCR product gel purification ............................................................11

2.6 Subcloning .................................................................................................................. 11
2.6.1
2.6.2
2.6.3
2.6.4

pBluescript ........................................................................................................................11
Plasmid DNA Extraction ..................................................................................................14
pBluescript + 1218 Restriction Digest .............................................................................14
PCR Amplification of Plasmid DNA ................................................................................15

3

RESULTS AND DISCUSSION...................................................................................... 15
3.1 Genomic DNA isolation............................................................................................. 15
3.2 PCR amplification ..................................................................................................... 16
3.3 Characterization of plasmids ................................................................................... 19
3.4 Gel Purification of ISGA 1222 & 1218 .................................................................... 21
3.5 Blunt-end ligation of ISGA 1218 into pBluescript ................................................. 23
3.6 Release of ISGA 1218 fragment from pBluescript ................................................. 23
3.7 PCR amplification of insert within pBluescript ..................................................... 25

4

CONCLUSION AND FUTURE WORK....................................................................... 26

5

LITERATURE CITED ................................................................................................... 29

6

APPENDIX ...................................................................................................................... 31
6.1 Appendix A ................................................................................................................ 31
6.2 Appendix B ................................................................................................................. 35
6.3 Appendix C ................................................................................................................ 35
6.4 Appendix D ................................................................................................................ 36
v

6.5 Appendix E ................................................................................................................. 36

vi

LIST OF FIGURES
Figure 1 The first two steps in the proposed metabolic pathway for 6:2 fluorotelomer
sulfonate degradation by Gordonia sp. NB4-1Y, including the desuflonation reaction
putatively catalyzed by ISGA 1222 and 1218 (Adapted from Van Hamme et al.
2013). ............................................................................................................................. 2
Figure 2 NB4-1Y genomic DNA isolation, labeled 1 and 2 (1 % agarose gel, 35 minutes at
100 V). ......................................................................................................................... 16
Figure 3 Gradient PCR amplification of ISGA 1222 and ISGA 1218 using PCR primers
designed to induce a 5' EcoRI and 3' XbaI restriction site. Each lane contains 5 µL of
an individual PCR reaction which was subject to a different thermocycling
temperature between 60-70 °C in 5 µL 5X loading buffer. ......................................... 18
Figure 4 Colony PCR amplification of DH5α competent E. coli cells. Genomic NB4-1Y
DNA was used as a positive control to determine whether colony PCR amplification
of transformant cells was an accurate method to determine the insertion of ISGA 1218
and ISGA 1222 into pBS. Each lane contains 2.5 µL of an individual PCR reaction
which was subject to a different thermocycling temperature between 60-70 °C in 5 µL
5X loading buffer......................................................................................................... 19
Figure 5 Restriction digest of pMAL-c2 and pBS to characterize plasmids. .......................... 21
Figure 6 Gel purified ISGA 1218 and unsuccessful recovery of ISGA 1222. ........................ 22
Figure 7 Attempted release of ISGA 1218 from MCS of pBS using EcoRI and XbaI single
digests and a EcoR/XbaI double digest. Digests were performed on mini-prep DNA
samples (1, 3 and 5). A positive control of EcoRV linearized pBS was used. ........... 24
Figure 8 Attempted excision of insert from pBS MCS by restriction digests using EcoRI and
XbaI. A single digest of EcoRI was performed, followed the removal of the enzyme
and its required buffers using the EZ-10 restriction enzyme removal kit. A second
single digest using XbaI was performed on the recovered EcoRI digested DNA. ...... 25
Figure 9 PCR amplification using ISGA 1218 specific primers on mini-prep DNA. Each lane
labeled 1-5 contains 5 µL of an individual PCR reaction containing template DNA
from pBS, the gel purified ISGA 1218 sample, and plasmid DNA from the five
recombinant colonies (listed in order after 1 Kb+ ladder)........................................... 26

vii

LIST OF TABLES
Table 1 Restriction digest performed on pBS and pMAL-c2 to characterize provided
plasmids. The single digests performed for pBS were: BsaI, and EcoRV, with a
double digest of BsaI and EcoRV. The single digest performed for pMAL-c2 were
BsaI, PstI and EcoRV, with double digests between BsaI and PstI, and BsaI and
EcoRV. .......................................................................................................................... 7
Table 2 Primer sequences for PCR amplification of the nucleotide sequences coding for the
proteins ISGA 1222 and ISGA 1218, including Tm values, length, GC content, and
melting temperature. ...................................................................................................... 9
Table 3 PCR reaction setup using the Q5 High-Fidelity DNA Polymerase kit. ....................... 9
Table 4 Thermocycling conditions for PCR amplification of the nucleotide sequences coding
for ISGA 1222 and ISGA 1218 in Gordonia sp. NB4-1Y. ......................................... 10
Table 5 Colony PCR reaction setup the Q5 High-Fidelity DNA Polymerase kit. .................. 11
Table 6 Restriction enzyme digest of pBS using the restriction enzyme EcoRV to allow for
linearization of the plasmid to insert gel purified ISGA 1218 sequence. .................... 12
Table 7 Ligation reactions prepared. ....................................................................................... 12
Table 8 Miniprep restriction digest to release insert from pBS MCS. .................................... 14
Table 9 Expected fragment size of each restriction digest of pMAL-c2 and pBS. ................. 20
Table 10 Transformation efficiency of control reactions and ISGA 1218 + pBS experimental
reaction. ....................................................................................................................... 40

viii

1

INTRODUCTION
The use of aqueous film-forming foams (AFFFs) to fight forest fires, and as fire

suppressants by the military and aviation industry, has resulted in their large-scale
deployment across natural ecosystems, resulting in the contamination of groundwater (Place
& Field 2012). Detectable levels of perfluorinated organic surfactants that are important
components of AFFFs have been reported in the livers of fish species found downstream of
areas where AFFFs have been used (Oakes et al. 2009). AFFFs are known to contain
perfluorinated organosulfur compounds that are not naturally metabolized and, as such, the
deployment of AFFFs has the potential to result in bioaccumulation of fluorinated
compounds within natural habitats.
It is well known that organic sulfonates are important sources of sulfur for soil
microorganisms, organisms that contribute greatly to the biogeochemical cycling of sulfur
(Boden et al. 2011, Li et al. 2013). The sulfur cycle allows for the movement of sulfur
through living organisms, minerals and the atmosphere. The cycling of sulfur is vital for
living organisms, as sulfur is a common component of proteins, cofactors and vitamins.
As the organosulfur compounds present within AFFFs, for example 6:2 fluorotelomer
sulfonate (6:2 FTS), are not found naturally and possess a backbone stabilized by strong
carbon-fluorine bonds, they are difficult to biodegrade, generating a need for bioremediation
tools for ecosystem restoration. Gordonia sp. NB4-1Y was isolated in Kamloops, British
Columbia from bis-(3-pentafluorophenylpropyl)-sulfide enriched vermicompost, and has
been found to metabolize a broad spectrum organosulfur compounds including 6:2 FTS (Van
Hamme et al. 2013). Gordonia spp. are attractive for bioremediation as they have broad
1

capacity for metabolism of natural and anthropogenic organics (Arenskotter et al. 2004).
Whole-genome shotgun sequencing revealed that the NB4-1Y genome possesses 72
monoxygenases and 52 dioxygenases. Additionally, using two-dimensional differential in-gel
electrophoresis, two putative nitrilotriacetate monoxygenases (NtaA), ISGA 1222 and 1218,
were found to be differentially upregulated when NB4-1Y was supplied 6:2 FTS as the sole
sulfur source instead of MgSO4. From this, it was hypothesized that these two enzymes may
be responsible for the initial desulfonation of 6:2 FTS in the proposed metabolic pathway
developed by Van Hamme et al. (2013) (Figure 1).

Figure 1 The first two steps in the proposed metabolic pathway for 6:2 fluorotelomer
sulfonate degradation by Gordonia sp. NB4-1Y, including the desuflonation reaction
putatively catalyzed by ISGA 1222 and 1218 (Adapted from Van Hamme et al. 2013).

This study is not the first to uncover the degradation of fluorinated sulfonates,
however it is one of few studies working with pure culture. Presently, the majority of
literature focuses on the microbial degradation of per- and poly- fluoroalkyl substances
(PFAS) by mixed cultures (Dinglasan et al. 2004; Rhoads et al. 2008; Wang et al. 2005). As
a result, there is a gap in knowledge relating to the biochemistry of microbial metabolism of
2

such compounds by pure cultures. Currently, only three studies have independently reported
microbial metabolism of PFASs by pure culture of Pseudomonas species, with only one
examining sulfur containing PFASs. Key et al. (1998) found Pseudomonas sp. strain D2 was
capable of using difluoromethane sulfonate, trifluoromethane sulfonate and 6:2 fluorotelomer
sulfonate [6:2 FTS; F(CF2)6CH2CH2SO3H] as sulfur sources for growth. In a different study
conducted by Wang et al. (2011) using sewage inocula, 6:2 FTS was found to be degraded to
5:3 fluorotelomer acid [5:3 acid; F(CF2)5CH2CH2CO2H], perfluorohexanoic acid [PFHxA;
F(CF2)5CO2H], perfluoropentanoic acid [PFPeA; F(CF2)4CO2H] and perfluorobutanoic acid
[PFBA; F(CF2)3CO2H] via 6:2 FTUCA [F(CF2)5CF5 = CHCO2H], 5:2 ketone
[F(CF2)5C(O)CH3] and 5:2 sFTOH [F(CF2)5CH(OH)CH3].

From this, Wang et al.

hypothesized the initial step of the microbial metabolism of 6:2 FTS by sewage inocula was
catalyzed by an unidentified bacterial alkane sulfonate-α-hydroxylase.
To determine the function of NtaA monoxygenases found in various bacterial
systems, researchers have previously reported using expression vector systems. Kim et al.
(2006) determined the function of an NtaA found in Corynebacterium glutamicum by cloning
and then expressing the gene in E. coli prior to purification. They first amplified the proteincoding region for the monoxygenase from chromosomal DNA by PCR using primers that
introduced restriction enzyme sites to the 5' and 3' ends before directional cloning into a
pPosKJ expression vector. The expression vector functioned to introduce a hexahistidine tag
and bacterial haemoglobin at the N-terminus of the NtaA protein, allowing for the
purification of the translated protein from harvested recombinant E. coli cell lysate through
an Ni-NTA column. The eluted fraction was then loaded onto a HiTrap Q anion exchange
column, and the bound proteins were eluted, prior to removing the hexahistidine tag removed
3

from the target protein with a recombinant tobacco etch virus protease. Finally, the proteins
were passed through a Ni-NTA column to remove the cleaved hexahistidine tags. Once
purified, Kim et al. performed in vitro NAD(P)H oxidoreductase assays for enzyme activity
characterization studies (Eichhorn et al. 1999).
The goal of this project was to clone the sequences encoding the two NtaA
monooxygenase proteins from NB4-1Y, namely ISGA1222 and 1218, into competent DH5α
E. coli to allow for future work to understand the role of the two proteins in the microbial
metabolism of 6:2 FTS. The cloning procedure used was similar to that described by Kim et
al. (2006) in that PCR primers were designed to add restriction enzyme sites to PCR products
to allow for directional cloning into the pMAL-c2 expression vector polylinker. Specifically,
an EcoRI restriction enzyme site was added to the 5' end by the forward primer, and an XbaI
site was added to the 3' end by the reverse primer. PCR amplicons were identified by size
comparison to a molecular weight marker, and were gel purified using a Qiagen II gel
extraction kit. The ISGA 1218 amplicon was used for sublconing into a pBluescript vector,
which will allow for sequencing of the inserted fragment. Future work with this project will
be to express the two NtaA proteins, ISGA 1218 and 1222, using the pMAL-c2 expression
vector.

Successful incorporation of the protein-coding regions for each protein in the

expression vector will result in the addition of a N-terminal maltose binding protein, which
like the hexahistidine tag used by Kim et al. (2006) will allow for the purification of the
target proteins through affinity chromatography. Once purified, the two NtaA enzymes can
be used for in vitro oxidoreductase assays monitoring the rate of NAD(P)H oxidation to
determine their role in 6:2 FTS desulfonation.

4

2

MATERIALS AND METHODS

All materials were purchased from Invitrogen unless otherwise stated.
2.1

Bacterial Strains and Growth Conditions
A stock culture of Gordonia sp. NB4-1Y was prepared from a CyroBank bead

harbouring NB4-1Y stored at -80 °C (blue cap, labeled NB4-1Y from sample June 2-14).
Using a sterilized loop, the CyroBank bead was used to streak for isolated colonies on
nutrient agar. The plate was incubated at 30 °C for four days, after which the plate was
sealed using Parafilm to prevent water loss and placed at 4°C.
2.2

DNA Extraction
Two 10 mL liquid nutrient broth cultures were aseptically inoculated using an isolated

colony from the prepared stock culture of NB4-1Y. The broth cultures were incubated at 30
°C for three days on a tube roller and the typical clumped white growth characteristic of
NB4-1Y was observed.

The liquid nutrient broth NB4-1Y cultures were then used to

inoculate YEME broth (Appendix B) cultures at 2 % v/v prior to incubation at 30 °C with
rotation for seven days.
Cells from each YEME culture (6 mL total) were harvested in microfuge tubes by
centrifugation (21 000 rpm, 5 minutes) and a portion of the original culture was used to check
for culture purity by streaking onto nutrient agar. The collected pellets were washed twice in
10.3 % sucrose and then resuspended in 500 µL filter sterilized lysozyme buffer (10 mg/mL)
including RNAse (50 µg/mL) in TE25S (1.5 M Tris-HCl, 0.5 M EDTA, 0.5 M sucrose, pH
8.0). Tubes were incubated for 1 hour in a 37 °C water bath and vortexed for 10 seconds
every 5 minutes.

5

Following incubation, 250 µL of 4 % SDS (Sigma-Aldrich) was added to each tube,
and tubes were then vortexed vigorously. With the addition of SDS into the lysozyme and
RNase resuspended cells, a white precipitate appeared in each tube.

In an attempt to

resuspend the precipitate in each tube, the tubes were heated for 2 minutes in the 37 °C water
bath. To remove protein contaminants, 250 µL neutral phenol:chloroform was added and
extracted (21 000 rpm, 5 minutes). The upper aqueous layer was transferred into a clean
microfuge tube and an additional volume of 250 µL neutral phenol:chloroform was added
and extracted (21 000 rpm, 5 minutes). This process was repeated three times. An equal
volume of chloroform (500 µL) was added and extracted to remove all phenol present in the
sample. This process was repeated a second time, with a volume of 300 µL. A total of 0.1
volumes of 3 M sodium acetate (30 µL, pH 4.8-5.1) and 2 volumes of ethanol (600 µL) were
added to each tube followed by gentle mixing by inversion to collect the fibrous DNA pellet.
Each pellet was transferred into a 70 % ethanol wash. Pellets were collected (21 000 rpm, 5
minutes) and the ethanol was removed to allow the pellet to air dry. Each pellet was
resuspended in 50 µL TE buffer (pH 8.0) and was stored at 4 °C.
2.3

DNA Quantification
The concentration of extracted DNA samples were quantified using a broad-range

Qubit Fluorometric DNA Quantification assay.
2.4

Characterizing plasmids

Plasmids pBluescript (pBS) and pMAL-c2 were obtained from Agilent Technologies and
New England BioLabs, respectively. Restriction digests were performed on pBS and pMALc2 to determine the quality of the provided samples. To determine which restriction enzymes
to use to digest the plasmids, published restriction maps were consulted (New England
6

BioLabs, Addgene). Based on the published restriction sites (Appendix C, Appendix D),
pMAL-c2 was digested with BsaI, PstI and EcoRV, and pBS was digested by BsaI and
EcoRV to generate fragments of each plasmid of known size, to be used to characterize and
identify each plasmid accurately (Table 1). Each reaction was incubated at 37 °C for 45
minutes, and the resulting DNA fragments were separated on a 1 % agarose gel for 1 hour.
The sizes of the generated restriction fragments were compared to a λ- HindIII molecular
weight marker.
Table 1 Restriction digest performed on pBS and pMAL-c2 to characterize provided
plasmids. The single digests performed for pBS were: BsaI, and EcoRV, with a double
digest of BsaI and EcoRV. The single digest performed for pMAL-c2 were BsaI, PstI
and EcoRV, with double digests between BsaI and PstI, and BsaI and EcoRV.
Digest
component
DNA
NEB 2 buffer
dH2O
Enzyme
Total volume:

pBluescript
Single Digest
Double Digest
66 ng/µL
66 ng/µL
3 µL
3 µL
22.7 µL
21.2 µL
3 units/µL
3 units/µL
30 µL
30 µL

2.5

Polymerase Chain Reaction Amplification

2.5.1

PCR Primer Design

pMAL-c2
Single Digest
Double Digest
66 ng/µL
66 ng/µL
3 µL
3 µL
18.8 µL
17.3 µL
3 units/µL
3 units/µL
30 µL
30 µL

Two sets of primers were designed for the amplification of genomic sequences
encoding for the proteins ISGA 1222 and ISGA 1218. Restriction enzymes were chosen
based on the presence of the restriction enzyme recognition site within the multiple cloning
site (MCS) of pBS and pMAL-c2. For directional cloning, the order of the restrictions
enzymes (5' or 3') was determined based on the order of the recognition sequences within the
pMAL-c2 MCS. Each primer was diluted to a final stock concentration of 100 µM with TE
buffer (pH 8.0) and a working solution concentration of 10 µM in TE buffer (pH 8.0).
7

The first set of primers designed for the nucleotide sequence coding for the protein
ISGA 1222 included different restriction enzyme recognition sites at the 5' end than the 3' to
allow for directional cloning into pMAL. For the forward primer F1222EcoLS, an EcoRI
site (GAA TTC) was added to the 5' end. An additional three-nucleotide extension (GAC)
was added to the 5' end to facilitate enzymatic digestion (New England BioLabs 2015). An
XbaI site (TCT AGA) was added to 5' end of the reverse primer R1222XbaLS, along with
three additional nucleotides (ACA). Following the XbaI site is the reverse compliment of a
stop codon TAG (CTA) to trigger the termination of transcription. The second primer set
was designed to clone with only one restriction site, XbaI, for non-directional cloning. The
forward primer F1222XbaLS included the addition of an XbaI site (TCT AGA) and a threenucleotide extension (GGT).
The primers designed for ISGA 1218 followed the same design guidelines as those
used for the design of the primers for ISGA 1222. For directional cloning, an EcoRI site
(GAA TTC) and a three-nucleotide extension (GTA) was added to the 5' end of the primer
and a XbaI restriction site (TCT AGA) including a three-nucleotide (TAC) extension added
to the 5' reverse compliment for the reverse primer. For non-directional cloning, an XbaI
forward primer was designed including the XbaI restriction site (TCT AGA) and a threenucleotide (ATG) extension. While designing the primers, the length, GC content, melting
temperature and 3' end stability were also considered. The sequences of the designed primers
are listed in Table 2.

8

Table 2 Primer sequences for PCR amplification of the nucleotide sequences coding for
the proteins ISGA 1222 and ISGA 1218, including Tm values, length, GC content, and
melting temperature.
Prime
FEcoRI1218
RXbaI1218
FEcoRI1222
r
Table
gtagaattcatgaacgta tactctagatcacgccgc gacgaattcatggctgat
Seque
aacgttg
ccccac
cgagag
nce
Tm
60 °C*
73 °C*
66 °C
Length
25
24
24
GC
conten
36%
62.5%
50%
t
Melt
52.7 °C
64.1 °C
57.5 °C
temp
* The Tm difference is greater than the recommended limit of 5 °C
2.5.2

RXbaI1222
acatctagactaaccggt
ccggcg
71 °C
24
58.3%
62.1 °C

PCR Reaction Setup
PCR reactions were performed using sterile PCR tubes and barrier tips in a biological

safety cabinet to reduce the possibility of contamination during reaction setup. Using a Q5™
High-Fidelity DNA Polymerase kit, reagents were added to nuclease-free dH2O in sterile
PCR tubes in the order outlined in Table 3.
Table 3 PCR reaction setup using the Q5 High-Fidelity DNA Polymerase kit.
Component
50 µL Reaction
Final Concentration
5x Q5 Reaction Buffer
10 µL
1x
10 mM dNTP
1 µL
200 µM
10 µM Forward primer
2.5 µL
0.5 µM
10 µM Reverse primer
2.5 µL
0.5 µM
Template Genomic DNA
0.5 µL (100 ng/ µL)
< 1000 ng
Q5 High Fidelity DNA Polymerase
0.5 µL
0.02 Units/ µL
Nuclease-free dH2O
Up to 50 µL
A 50 µL reaction was prepared to amplify both ISGA 1222 and ISGA 1218 for each
temperature of the temperature gradient (60-70 °C), and a negative control containing no
DNA in the reaction was prepared for each sequence of interest. The samples were vortexed
9

for 5 seconds before they were placed in a 98 °C preheated thermocycler. The thermocycling
conditions for the gradient PCR reaction is listed in Table 4.
Table 4 Thermocycling conditions for PCR amplification of the nucleotide sequences
coding for ISGA 1222 and ISGA 1218 in Gordonia sp. NB4-1Y.
ISGA 1218
ISGA 1222
Temperature (°C) Time (min) Temperature (°C) Time (min)
98
2
98
2
Initial denaturation
98
0.5
98
0.5
60-70
0.5
60-70
0.5
35 cycles
72
1.5
72
1.5
Final Extension
72
2
72
2
Hold
4
∞
4
∞
Following amplification, PCR products were visualized on a 2 % agarose gel (90
volts, 3 hours). PCR products were quantified by comparison to a 1 Kb plus DNA ladder.
2.5.3

Colony PCR Amplification of E. coli (DH5α) Using NB4-1Y Specific Primers

A colony PCR reaction was set up to determine whether the primers designed to amplify
ISGA 1222 and ISGA 1218 in Gordonia sp. NB4-1Y amplify any genes present in E. coli
DH5α. An isolated colony of DH5α was transferred via a sterile toothpick into 30 µL
nuclease-free water and was subject to 70 ̊C in a thermocycler for 10 minutes to lyse the
cells. During the cell lysis, PCR reaction tubes were prepared containing PCR reactions to a
total volume of 25 µL (Table 5). Following the completion of the cell lysis, 1 µL of the lysed
solution was added to each reaction, and was subject to PCR amplification following the
temperature gradient previously described (Table 4). A positive control was prepared using
extracted NB4-1Y DNA.

10

Table 5 Colony PCR reaction setup the Q5 High-Fidelity DNA Polymerase kit.
Component
25 µL Reaction
Final Concentration
5x Q5 Reaction Buffer
5 µL
1x
10 mM dNTP
0.5 µL
200 µM
10 µM Forward primer
1.25 µL
0.5 µM
10 µM Reverse primer
1.25 µL
0.5 µM
Template Genomic DNA
1 µL (Lysed DH5α)
< 1000 ng
Q5 High Fidelity DNA Polymerase
0.25 µL
0.02 Units/ µL
Nuclease-free dH2O
Up to 25 µL
2.5.4

ISGA 1222 & 1218 PCR product gel purification
To isolate the ISGA 1218 PCR product from the non-specific amplicons obtained

through PCR amplification, DNA fragments were separated on a 1% agarose gel (90 V, 45
minutes) prior to using a Qiaex II gel extraction kit. To ensure maximum recovery, 15 µL of
the PCR product was loaded with 5 µL 5X loading buffer. Bands were visualized using a
GelDoc system (BioRad), and the 1473 bp band corresponding to the ISGA 1218 gene was
excised using a scalpel and placed into a weighed tube. The Qiaex II gel extraction kit
protocol was followed as per the manufactures instructions (Qiagen). The pelleted DNA was
resuspended in TE buffer (20 µL, pH 8.0) to elute the DNA into solution. The eluted DNA
was then transferred into a new tube, and a second elution was performed. The gel purified
first and second DNA elutions were visualized following separation on a 1 % agarose gel at
90 V for 45 minutes.
2.6

Subcloning

2.6.1

pBluescript
The restriction enzyme EcoRV (5'-GAT|ATC-3') was used to linearize pBS to allow

for the blunt-ended ligation of the gel purified ISGA 1218 amplicons into the linearized

11

plasmid. The digest was prepared as per Table 6, and was incubated at 37 °C for 1 hour. The
enzyme was then deactivated at 80°C for 20 minutes.
Table 6 Restriction enzyme digest of pBS using the restriction enzyme EcoRV to allow
for linearization of the plasmid to insert gel purified ISGA 1218 sequence.
Digest Component Volume Concentration
pBluescript
2.8 µL
66 ng/µL
NEB 3 buffer
3 µL
1X
dH20
22.7 µL
EcoRV
1.5 µL
15 units
TOTAL
30 µL
-

Following the deactivation of the enzyme, ligation reactions were prepared (Table 7).
Table 7 Ligation reactions prepared.
Ligation Reaction

i
ii
iii
iv
v
10 ng 10 ng
100 ng
100 ng
pBluescript
100 ng linearized
uncut uncut linearized linearized
ISGA 1218
10 µL
5X Ligation Buffer
2 µL
2 µL
2 µL
2 µL
T4 DNA Ligase
1 µL
1 µL
1 µL
dH20
29 µL 21 µL 21.5 µL
20.5 µL
10.5 µL

The four control reactions used in the transformation experiment were prepared as
follows:
i.

10 ng uncut pBS (no ligation reaction). From this transformation, the maximum
efficiency of the DH5α competent cells can be determined.

ii.

10 ng uncut pBS (ligation reaction). The addition of DNA ligase should reduce
the transformation efficiency.

iii.

100 ng linearized pBS (no DNA ligase). From the EcoRV restriction digest of
pBS, 100 ng of linearized pBS was used to demonstrate the efficiency of
12

degradation. As linear DNA is degraded by exonucleases in a bacterial cell, if the
digestion by EcoRV was incomplete, the uncut covalently, closed, circular (ccc)
pBS will result in recombinant colonies and blue colonies will be visible.
iv.

100 ng linearized pBS (ligation reaction). By adding DNA ligase to the linearized
pBS, the ability of the recircularization can be identified.
To allow for the blue-white α-complementation test to detect the successful insertion

of an insert into the MCS of pBS, X-gal (20 mg/mL in dimethylformamide) and isopropyl βD-1-thiogalactopyranoside (IPTG, 20 mg/mL in dH2O) were added (40 µL each) and spread
using spread plating techniques onto ampicillin (100 mg/L) containing nutrient agar plates
and were incubated at 37 °C until the liquid evaporated. During this time, DH5α competent
cells were removed from the -70 °C freezer and thawed on ice. All transformations were
prepared on ice, adding 2.5 µL of either control or experimental ligation reactions into chilled
tubes. The thawed competent cells were added to allow for proper dispersal of the ligation
reaction with minimal mechanical damage to the fragile competent cells. The competent
cells and ligation reaction mix was then incubated for 30 minutes on ice. After incubation,
the competent cells were heat shocked at 42 °C for 2 minutes. The competent cells were
returned back onto ice, and 1 mL of LB media was added. The solution was placed in the 37
°C incubator for 30 minutes. After incubation, 100 µL of each transformation reaction was
aseptically spread onto agar plates containing ampicillin, X-gal and IPTG. The plates were
incubated at 37 °C for 24 hours. The number of blue and white colonies on each plate was
recorded to determine transformation efficiency.
Successful transformants were identified as white colonies through the αcomplementation test. Using a sterile loop, transformants were streaked for isolated colonies
13

on agar plates containing ampicillin, X-gal, and IPTG. Plates were incubated for 24 hours at
37 °C.
2.6.2

Plasmid DNA Extraction
Plasmid minipreps were prepared using the Qiagen plasmid purification system.

Isolated transformant colonies were used to inoculate 4 mL liquid broth tubes containing
ampicillin (100 mg/L) prior to incubation for 12 hours at 37 °C while shaking. The plasmid
minipreps were prepared as per the protocol described by the Molecular Cloning Handbook.
The products were visualized on a 1 % agarose gel (100 V, 45 minutes).
2.6.3

pBluescript + 1218 Restriction Digest
To release the insert from the MCS of pBS, EcoRI and XbaI restriction digests were

performed on the plasmid DNA. The PCR primers used to amplify ISGA 1218 gene were
designed to introduce a 5' EcoRI site and a 3' XbaI site, and as such, the first restriction digest
performed included single digests by EcoRI and XbaI to linearize the DNA and a double
digest of EcoRI and XbaI to release the ISGA 1218 fragment from the pBS vector (Table 8).
The digests were incubated at 37 °C for 1 hour, and were visualized on a 1 % agarose gel
(100 V, 45 minutes).
Table 8 Miniprep restriction digest to release insert from pBS MCS.
Digest component
DNA
Neb 2
dH2O
Enzyme
TOTAL

pBluescript + ISGA 1218
Single Digest
Double Digest
Concentration
2 µg
2 µg
66 ng/µL
3 µL
3 µL
1X
22.7 µL
21.2 µL
1.5 µL
1.5 µL (of each enzyme) 15 units (of each enzyme)
30 µL

30 µL

-

14

After the insert was not observed to be released from the pBS MCS, a second
restriction digest was performed. As the insert was introduced into the EcoRV blunt-ended
site of the pBS MCS, restriction sites were examined on either side of the EcoRV site in
attempt to excise the insert. Using this technique, HindIII and XbaI were used. Additionally,
instead of performing two single digests and a double digest including the two restriction
enzmyes, a HindIII single digest was performed, and the restriction enzymes and buffers
were removed using the EZ-10 restriction enzyme cleanup protocol before the second
restriction enzyme digest by XbaI.

The EZ-10 protocol was followed as per the

manufacturer’s instruction (Bio-Basic). The restriction digests and EZ-10 products were
visualized on a 1% agarose gel (100 V, 45 minutes).
2.6.4

PCR Amplification of Plasmid DNA

The plasmid DNA samples were subject to PCR amplification using the ISGA 1218
directional primers (5' EcoRI site, 3' XbaI site) used to generate the amplified sequence
transformed into pBS. A control sample was run using pBS with no insert as the DNA
template to see if the primers amplified any sequence within pBS.

Following PCR

amplification, the PCR products were observed on a 1 % agarose gel (100 V, 45 minutes).

3

RESULTS AND DISCUSSION

3.1

Genomic DNA isolation
In order to isolate genomic DNA from NB4-1Y to be used as a template for PCR

amplification of the target genes, a modified Streptomyces DNA extraction protocol was used
(Kieser et al. 1985). Generally, liquid cultures were grown for 10 days prior to extractions as
Gordonia and related species have slower growth rates than E. coli (Fusconi et al. 2006;
15

Marr 1991). In addition, a high sucrose medium was used as it is believed that high osmotic
stress weakens the robust cell wall of these organisms (Van Hamme, personal
communication, September 2015) to allow for more efficient lysis. DNA (100 ng/µL) was
recovered from 6 mL cultures, and gel electrophoresis revealed high quality, high molecular
weight DNA free from RNA contamination (Figure 2). With this DNA in hand, it was
possible to carry out PCR reactions.

1 Kb +
(175 ng)

NB4-1Y
genomic DNA
1

2

Figure 2 NB4-1Y genomic DNA isolation, labeled 1 and 2 (1 % agarose gel, 35 minutes
at 100 V).
3.2

PCR amplification
PCR reactions were performed to amplify the nucleotide sequences of interest for each

NtaA enzyme to subsequently transform into a pBS cloning vector.

The Q5 High Fidelity

DNA Polymerase was selected for this, as the PCR products formed are blunt-ended (New
England BioLabs), allowing for ligation into pBS without causing a frameshift. After taking
into account all of the various factors when designing the primers including the length, GC
content, Tm values and 3' end stability, the ISGA 1218 directional primer pair (5' EcoRI, 3'
XbaI site) exceeded the suggested 5 °C Tm difference; the forward primer had a Tm value of
16

60 °C, whereas the reverse primer had a Tm value of 73 °C (New England BioLabs). With
this large difference in Tm values, the suggested annealing temperature was set at 63 °C.
The ISGA 1222 directional primer pair (5' EcoRI, 3' XbaI site) did not exceed this difference,
and was suggested to anneal at 69 °C. As each target annealing temperature was found
between 60 and 70 °C, a gradient PCR was performed for amplification of each target in an
attempt to determine which temperature provided the greatest amount of product (Figure 3).
When examining PCR products for each target, ISGA 1222 and 1218, several nonspecific products were visualized under each target band, which may have been attributed to
the concentration of the polymerase, the length of the extension time, or the annealing
temperature (New England BioLabs). This protocol was repeated using increased
concentrations of the template DNA (10 ng, and 100 ng) into the PCR reaction mixture;
however the non-specific banding was found to increase with increasing concentration of the
genomic DNA template.

17

1 Kb +
(175 ng)

ISGA 1222

ISGA 1218

1 Kb +
(175 ng)

1347 bp
1473 bp

Figure 3 Gradient PCR amplification of ISGA 1222 and ISGA 1218 using PCR primers
designed to induce a 5' EcoRI and 3' XbaI restriction site. Each lane contains 5 µL of
an individual PCR reaction which was subject to a different thermocycling temperature
between 60-70 °C in 5 µL 5X loading buffer.
Once the PCR conditions were optimized to amplify products of approximately the
same size as the desired products, a colony PCR amplification was performed to determine
whether any sequences in E. coli would be amplified using these primers. It was thought that
if E. coli did not have any sequences amplified by the primers, then a colony PCR
amplification of the transformant cells after subcloning would be a suitable method of
determining whether the desired insert was ligated into pBS. E. coli DH5α cells were used
for a temperature gradient PCR and, as seen in Figure 4, there is relatively weak
amplification of several targets within E. coli using primers for ISGA 1222 and 1218 at all
temperatures except (61.2 ºC).

At this temperature, not only were there three bands with

high concentrations of PCR products, but also one band was approximately the same size as
the each target (approximately 1400 bp), and as such this method of determining the presence
of the insert was not used.

However, with the successful amplification of each target
18

sequence, the plasmids for which the sequences would later be transformed were
characterized to confirm their identity.

1 Kb + NB4(175 ng) 1Y

ISGA 1222

ISGA 1218

NB41Y

Figure 4 Colony PCR amplification of DH5α competent E. coli cells. Genomic NB4-1Y
DNA was used as a positive control to determine whether colony PCR amplification of
transformant cells was an accurate method to determine the insertion of ISGA 1218 and
ISGA 1222 into pBS. Each lane contains 2.5 µL of an individual PCR reaction which
was subject to a different thermocycling temperature between 60-70 °C in 5 µL 5X
loading buffer.
3.3

Characterization of plasmids
To confirm the identity of the supplied plasmids, pBS and pMAL-c2, the plasmids

were characterized using restriction digests by restriction enzymes to compare to published
restriction maps (NebCutter) (Appendix C, Appendix D). The restriction enzymes chosen

19

yielded fragments that could be detected using a 1 % agarose gel, and provided characteristic
banding patterns that were used to confirm the purity and authenticity of each plasmid.

Table 9 Expected fragment size of each restriction digest of pMAL-c2 and pBS.
pMAL-c2 (6643 bp)
Restriction Digest Expected fragment size (bp) Observed fragment size (bp)*
BsaI
4101, 1480, 1067
7000, 6000, 3000, 2000
PstI
6643
6700
EcoRV
6643
6700
BsaI + PstI
4101, 1132, 1067, 348
3300, 1400, 1000
BsaI + EcoRV
3386, 1480, 1067, 710
3300, 1400, 1000, 700
pBluescript (2958 bp)
Restriction Digest Expected fragment size (bp) Observed fragment size (bp)*
BsaI
2958
3000
EcoRV
2958
3000
BsaI + EcoRV
1410, 1188
1400, 1300
When analyzing the restriction digest for pMAL-c2 (Table 9) the PstI and EcoRV
digest resulted in the expected linearized 6643 bp fragment characteristic for this plasmid.
The BsaI single digest was inconclusive, however the BsaI/PstI digest provided four bands
approximately the same size as the expected fragments (4101, 1132, 1067, 348bp). Further
support for the characterization of pMAL-c2 was provided from the BsaI/EcoRV digest,
containing all four expected fragments (3386, 1480, 1067, 710 bp). The combination of the
results from the single and double digests allowed for the conclusion of a pure sample of
pMAL-c2.
The restriction digests for pBS showed that BsaI and EcoRV single digests resulted in
the expected linearized 2958 bp fragment, and the BsaI/EcoRV double digest provided two
bands approximately the same size as the expected 1410 and 1188 bp fragments (Figure 5).
When looking at the BsaI/EcoRV digest, two additional bands can be seen above the two
digested fragments. The larger of the two bands is a result of an incomplete digest, and the
20

band is remaining ccc pBS, whereas the second band, directly above the two digested
fragments is a result of a partial single digest by either BsaI or EcoRV. The confirmed
identity of each plasmid through restriction enzyme characterization allowed the progression
to subcloning the PCR amplified sequences, first into pBS.
1 Kb + BsaI
(175 ng)

PstI

EcoRV BsaI + BsaI + BsaI EcoRV BsaI +
EcoRV
PstI EcoRV

pMAL-c2

pBluescript

Figure 5 Restriction digest of pMAL-c2 and pBS to characterize plasmids.
3.4

Gel Purification of ISGA 1222 & 1218
Before subcloning into pBS, gel purification was performed due to the high number of

non-specific PCR products. By selectively gel purifying the desired product from the nonspecific PCR products, a concentrated sample of each target amplicon could be ligated into
pBS without competition with unwanted DNA fragments in solution.

The Qiagen

purification protocol used relies on agarose solubilization followed by the selective
absorption of the QiaxII silica beads to the nucleic acids in high salt concentrations provided
by the QXI buffer (7M NaPO4, 10 mM NaAc).

21

1 Kb +
(175 ng)

ISGA 1222

ISGA 1218

Figure 6 Gel purified ISGA 1218 and unsuccessful recovery of ISGA 1222.
Unfortunately, the ISGA 1222 amplicon was not successfully purified (Figure 6),
which can likely be attributed to insufficient binding of the nucleic acids to the QIAEX II
silica beads. If the nucleic acids did not bind to the silica beads, DNA would have been
discarded in the supernatant. The ISGA 1218 amplicon was successfully recovered, although
two bands were seen on the gel due to imprecise excision of the desired band before gel
purification. If the procedure were to be repeated, a more precise excision of the band form
the gel could have been achieved if the gel was run for an additional 20 minutes, allowing
more time for the bands to separate from one another. Despite the presence of two bands, the
sample was quantified (5 ng/µL) by comparison to the 1 Kb + DNA ladder and used in
subsequent transformation experiments as the lower band, corresponding to ISGA 1218, was
a higher concentration than the non-target amplicon.

22

3.5

Blunt-end ligation of ISGA 1218 into pBluescript
The gel purified ISGA 1218 amplicon was ligated into a pBS cloning vector with the

intention of later sequencing the insert to compare to the published sequence data for ISGA
1218. The ISGA 1218 amplicon was ligated into a blunt end EcoRV digested pBS to ensure
the insertion of the fragment would not cause a frameshift. The ligation products were then
transformed into E. coli DH5α cells based on the high transformation efficiency of the strain
(Taylor et al. 1993). Following incubation, five recombinant white colonies were observed
demonstrating the successful transformation of the ISGA 1218 insert into pBS. With the
identification of recombinant clones, it was possible to extract the plasmid DNA for further
analysis to confirm the presence of the insert.
3.6

Release of ISGA 1218 fragment from pBluescript
Plasmid DNA of the ISGA 1218 + pBS transformants was isolated using a plasmid

mini-prep DNA isolation, and was used to excise the ISGA 1218 fragment from the MCS of
pBS. A blunt-end transformation of the fragment into pBS was used to allow for the EcoRI
and XbaI restriction sites introduced into the fragment by PCR amplification to remain intact.
By conserving their sequences, this would allow for restriction digest overhang at these sites,
resulting in the sticky ends allowing for directional cloning into the pMAL-c2 expression
vector. When the plasmid DNA was digested with EcoRI and XbaI (Figure 7), the plasmid
was only found to linearize, and no release of the ISGA 1218 fragment was observed. This
result suggested the restriction enzymes were only cutting at the sites present within the MCS
of pBS, and not digesting the restriction sites introduced into the ISGA 1218 fragment.

23

1 Kb + ccc
(175 ng) pBS

lin
pBS

+ control

ccc 1 EcoRI

XbaI

1

XbaI EcoRI ccc 5 EcoRI XbaI
+
XbaI

EcoRI ccc 3 EcoRI
+
XbaI

3

EcoRI
+
XbaI

5

Figure 7 Attempted release of ISGA 1218 from MCS of pBS using EcoRI and XbaI
single digests and a EcoR/XbaI double digest. Digests were performed on mini-prep
DNA samples (1, 3 and 5). A positive control of EcoRV linearized pBS was used.

A second attempt to excise the ISGA 1218 insert from pBS was performed using
EcoRI and XbaI single digests, separated by the removal of the restriction enzyme and their
associated buffers. The first single digest was performed using EcoRI, which was observed to
linearize the plasmid (Figure 7). Following the incubation of the digest, the restriction
enzyme and associated buffers were removed from the linearized plasmid using the EZ-10
Plasmid DNA purification kit. The DNA was selectively isolated in the silica gel-based
column, allowing for all of the unwanted products in the restriction digest solution to be
washed away. Following the removal of the products, the DNA was precipitated using
ethanol and eluted in TE buffer (pH 8.0), allowing for unhindered digestion of the linearized
plasmid by XbaI.
24

Unfortunately, the digestion of the linearized pBS + ISGA 1218 plasmid did not
result in the release of the insert; rather, no change in the nature of the plasmid was
visualized (Figure 8).

Figure 8 Attempted excision of insert from pBS MCS by restriction digests using EcoRI
and XbaI. A single digest of EcoRI was performed, followed the removal of the enzyme
and its required buffers using the EZ-10 restriction enzyme removal kit. A second
single digest using XbaI was performed on the recovered EcoRI digested DNA.

3.7

PCR amplification of insert within pBluescript
After being unable to release the insert from the MCS of pBS, a PCR amplification of

the inserted DNA fragment was performed to ensure the single fragment ligated into pBS
was a PCR product of the ISGA 1218 directional cloning primer pair. The PCR reaction
containing pBS as the DNA template did not result in any amplification, whereas the gel
purified ISGA 1218 fragment acting as the positive control produced two bands (Figure 9).
This result was already observed after gel purification of the fragment, having the upper band
corresponding to the desired 1473 bp ISGA 1218 fragment. The presence of two bands
decreases the chance of the desired band being ligated into pBS. However, when examining
25

the bands amplified within pBS, the amplified target is the same size as the desired band, and
did not contain additional non-specific PCR products. Although the insert was successfully
amplified using ISGA 1218 specific primers, because the insert was unable to be removed
from pBS, sequencing was not performed. Although the amplified ISGA 1218 sequence was
gel purified based on the fragment size, transformation assays consist of the movement of a
single molecule into a vector, such as pBS. As such, it is possible an undesirable, or misamplified PCR product was introduced into pBS, which resulted in the inability to excise the
ligated fragment from the vector.

Figure 9 PCR amplification using ISGA 1218 specific primers on mini-prep DNA. Each
lane labeled 1-5 contains 5 µL of an individual PCR reaction containing template DNA
from pBS, the gel purified ISGA 1218 sample, and plasmid DNA from the five
recombinant colonies (listed in order after 1 Kb+ ladder).
4

CONCLUSION AND FUTURE WORK
As the ISGA 1218 insert could not be excised from the MCS of pBS, in future studies a

restriction map could be generated from the transformant clone using restriction enzymes
whose sites are present in ISGA 1218 to determine if the target sequence was properly
26

amplified and successfully transformed into pBS. The inability to excise the insert from pBS
suggests errors in the amplification of the target sequence. As the purpose of first cloning the
insert into the pBS was to visualize the excision of the insert from pBS generating sticky
ends to allow for directional cloning, sequencing was not performed on the generated clones,
as the ligated insert is of limited use due to its inability to be excised.
For this project to continue, the PCR amplification of the nucleotide sequences coding
for ISGA 1218 and 1222 would need to be repeated to generate fresh, amplified targets to be
gel purified and used in a pBS transformation assay. Assuming the insertion of a target
sequence that can be excised from pBS using the restriction enzyme sites introduced on the 5'
and 3' end, the insert could be sequenced using 5' M13pUC-fwd and 3' T3 sequencing
primers. The sequencing data obtained can be compared to the sequence data published (see
Appendix C) to determine whether or not the sequences align. Any discrepancies in the
sequences could result in the misfolding of the protein and inhibit further processing once
ligated into the pMAL-c2 expression vector. If, however, the sequence data aligned, and the
insert could be digested from pBS, the excised insert could be gel-purified and ligated into an
EcoRI and XbaI digested pMAL-c2 expression vector to allow for directional,
complementary ligation of the insert into the vector.
Similarly to pBS, the blue-white α-complementation test is also used in the pMAL-c2
expression vector system (New England BioLabs) to determine the presence of an insert
within the MCS. In contrast to pBS, pMAL-c2 series also encodes for a maltose binding
protein by the malE gene, upstream of the MCS to be attached to the 5ʹ′ end of the ligated
insert. The deletion of the malE signal sequence in the –c2 variety of the plasmid results in
cytoplasmic expression of the fusion protein, eliminating the possibility of being exported
27

into the periplasm of the cell. Between the malE gene and the MCS of the vector, a spacer
sequence coding for 10 asparagine amino acid residues is present to insulate the maltose
binding protein from the protein of interest once translated within the cell. Once inserted into
the vector, and a white colony is recovered, the purification of the NtaA target proteins can
be achieved by generating a large volume of biomass to be subjected to amylose affinity
chromatography to selectively bind to the maltose binding protein + NtaA protein complex
from all of the other proteins present within E. coli. Once all of the unwanted proteins are
washed away from the column, the maltose binding protein + NtaA protein can be eluted
through a maltose wash. The purification of the NtaA proteins from the maltose binding
protein can then occur through proteolysis by a Factor-Xa protease, digesting at a cleavage
site present between the malE gene and the MCS, releasing the maltose binding protein from
the two NtaA proteins. From here subsequent assays determining the function of each NtaA
enzyme can be determined, furthering our understanding of the metabolism of 6:2 FTS by
Gordonia sp. NB4-1Y.

28

5

LITERATURE CITED

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Marr, AG. Growth rate of Escherichia coli. Microbiological Reviews. 55(2): 316-333 (1991).
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30

6

APPENDIX

6.1

Appendix A

ISGA 1222
DNA sequence
EcoRI
5ʹ′ GAC GAA TTC ATG GCT GAT CGA GAG 3ʹ′ F1222EcoLS
5ʹ′ ATG GCT GAT CGA GAG CTC CAT CTG GGC GTC AAT
GTC CTC TCG GAC GGT ATG CAC CCA GCC GCG TGG CAG TAT CCG
AGT TCC GAT CCG TCG TGG TTC ACG GAT CCG GCG TAC TGG ATT
CGT GTT GCG CAG ATC GCG GAG CGA GGA ACC CTC GAC GCG GTC
TTC CTC GCC GAC AGT CCG TCG TTG TTC CAG CCG CCC GAC CAG
CCG CTG AGT GCG CCA CCG TTG GCC CTG GAC CCG ATC GTG TTG
TTG TCG ACA CTG GCA TCG GTG ACC ACA CAC ATC GGA CTC ATC
GGT ACG GTG TCG ACC TCG TTC GAG GAG CCG TAC AAC GTC GCC
CGC CGA TTC TCG ACG CTG GAC CAC CTC AGC CGG GGT CGT GTG
GCA TGG AAC GTC GTG ACG AGT AGT GAT CGG TAT GCC TGG AAC
AAT TTC GGT GGT GGT GAA CAA CCC GAC CGC GCT ACT CGA TAC
GAG CGG GCC GGC GAG TTC ATC GAA GTC GTC CGG GCA TTG TGG
GAT TCG TGG GAC GAC GAC GCA GTT GTC GCC GAC AAG TCC ACG
GGT GCG TTC AGT AAG GTC GGT GCG ATA CGA CCG ATC CGG CAT
CGC GGT GGG CAC TTC TCG GTG GAC GGG CCG TTG ACT CTA CCC
AGA TCC CCA CAG GGG CAT CCG GTG TTG TTT CAG GCA GGC GGT
TCC ACC GGC GGG TTG GAT CTG GCG GCG AAG TAC GCC GAC GGG
GTC TTT GCG GCA CAG GCC TCG CTC GAG GAT GCG CTG TCC AAC
GCG CAG GAG CTG CGG AGT CGG TTG ATC GCG CAT GGC CGT CCC
31

GCC GAG GCG ATC CGA ATC ATG CCT GGC TTG TCG TTC GTG CTC
GGC AGT ACG GAG GCA GAG GCC AGG TCG CGA AAC GAC GAA TTG
AAC GAG CTC GCC GGG GAT CGA CGC CTG GCA CAT CTG GCT GGT
CAA CTC AGC GTC GAT GTG GCG GAG CTG AAG TGG GAC AAG CCG
CTT CCC GGT TGG CTC CTC GAG GGC GCG GCG CCG ATC AGC GGT
TCC CAG GGA GCT CGC GAC ATC GTC GTC AAC ATC GCT CGG CGG
GAG AAC CTG ACC GTG CGT CAG CTG CTC GAT CGG GTG ATC ACG
TGG CAC CGC TTC GTG GTC GGA TCG CCT GAA CAG ATC GCC GAT
GCC ATC GAG GAC TGG TTC GTT GCG GGC GCT GTC GAC GGC TTC
AAC CTG ATG CCG GAT GTC TTC CCG TCG GGT CTC GAG TTG TTC
GTC GAC CAC GTC GTA CCG ATC CTC CGG GAC CGA GGG TTG TTC
CGG CGG GAG TAC ACA TCG ACG ACA TTG CGT GGG CAT CTG GGC
CTC GAG CGC ACC CCA GAC CGG CCG TCG TCG GGT TCG ATC
CGC CGG ACC GGT TAG 3ʹ′
5ʹ′ GCG GCC TGG CCA ATC AGA TCT TAG 3ʹ′ R1222XbaLS
XbaI
Amino acid sequence
MADRELHLGVNVLSDGMHPAAWQYPSSDPSWFTDPAYWIRVAQIAERGTL 50
DAVFLADSPSLFQPPDQPLSAPPLALDPIVLLSTLASVTTHIGLIGTVST
100
SFEEPYNVARRFSTLDHLSRGRVAWNVVTSSDRYAWNNFGGGEQPDRATR 150
YERAGEFIEVVRALWDSWDDDAVVADKSTGAFSKVGAIRPIRHRGGHFSV 200
DGPLTLPRSPQGHPVLFQAGGSTGGLDLAAKYADGVFAAQASLEDALSNA 250
QELRSRLIAHGRPAEAIRIMPGLSFVLGSTEAEARSRNDELNELAGDRRL
300
AHLAGQLSVDVAELKWDKPLPGWLLEGAAPISGSQGARDIVVNIARRENL 350
TVRQLLDRVITWHRFVVGSPEQIADAIEDWFVAGAVDGFNLMPDVFPSGL 400
ELFVDHVVPILRDRGLFRREYTSTTLRGHLGLERTPDRPSSGSIRRTG

32

ISGA 1218
DNA sequence
EcoRI
5ʹ′ GTA GAA TTC ATG AAC GTA AAC GTT G 3ʹ′ F1218EcoLS
5ʹ′ ATG AAC GTA AAC GTT GTT GGC GGA ATC TCT
CAA TGG GAC ACT GTT CAG GCC GAT GCG TGG TGG TCG GCT GCA
GTC GGT CCA GCA ACG AAC GGA GAG ATC TCG ATG TCG GCA CAT
GGG CGT CCA ATT CAC TTG GGC GGG TTT TTG ATT GCA GGG AAT
GTA ACC CAC AGT CAT CCT TCG TGG CGT CAT CCA CGC AGT GAT
CCC GGG TTT CTC ACA CCG GAG TAC TAC CAG CAC CTC GGT AGG
GTT TTC GAA CGC GCG AAA TTG GAC TTC GTC TTC TTT GCC GAC
AAT TCT GCG ACT CCT GCC AGC TAC CGC AAC GAT ATT CGT GAC
CCG CTC GCT CGC GGT ACT CAG AGT GCA GCC GGG TTG GAT CCC
CGC TTC GTC GTT CCT GTC GTC GCG GGT GTC ACG CGC AAC CTG
GGG ATC GTA TCG ACC ACG TCG GCG ACG TTC TAC TCG CCA TAC
GAC CTC GCC CGG AGC TTT GCC ACT CTG GAT CAT CTG ACC CAC
GGC CGC GTC GGT TGG AAT GTT GTG ACT TCC AAT ACG ACC GTC
GAG GCG CAG AAC TTC GGG CTT GCC CGA CAC CTC GAC CAT GAC
GTG CGA TAC GAC CGT GCC GAG GAA TTG CTT GAG GTC GCG TTC
AGG CTG TGG GCC AGT TGG GAC GAT GGA GCT CTG ATC CAG GAT
AAA GAG GCG GGT GTC TTC GCC GAC CCG GAC CTG ATT CAC AGG
CTC GAT CAT CAC GGG GAG AAC TTC GAT GTT CGG GGC CCG CTG
TCG GTT CCC CGC TCA CCG CAG GGA CGG CCG GTC ATC TTT CAA
GCG GGA TCA TCC ACC CGC GGT CGG GAT TTT GCT GCG CGC TGG
GCA GAA GCG ATT TTC GAG ATC GAC CCG ACG TCT GTG GGG CGT
33

AAG GCC TAC TAC GAC GAC ATC AAG TCG CGA GCC TCC GAC TTC
GGT CGT GAT CCC GAC GGC GTC AAG ATA CTC CCG TCG TTC ATT
CCG TTT GTG GGT GAG ACC GAG TCG ATC GCA CGG GAA AAG CAG
GCG TTC CAC AAC GAA CTG GCC GAT CCG ACC GAT GGA TTG ATC
ACG CTG TCG GTG CAC ACC GAC CAT GAT TTC TCC GGC TAT GAC
CTC GAC GCT GTG ATC GCC GAC ATC GAT GTT CCA GGG ACG AAG
GGG CTT TTC GAA GTC GCT CGG AGT CTG AGT GTG AAC GAG AAC
CTG ACG CTG CGC GAT ATC GGA AAG CTG TAC GCC CAG GGC GTG
TTA TTG CCG CAG TTC GTG GGT ACC GCG GCT CAG GTG GCC GAC
CAG ATC GAG GCT GCC GTC GAC GGT GGA GAG GCT GAT GGG TTC
CTC TTT TCG GCC GGG TAT ACG CCT GGC GGA TTC GAG GAG TTC
GCC GAT CTC GTC ATC CCG GAA CTG CAG CGG CGA GGG CGG TTT
CGT ACG GAG TAC ACG GGT TCG ACG CTG CGT GAA CAT CTG GGT
CTA CCC GCT GAT GCG AAT CTT GTG CCC GTT CCG CGC AAG GCA
GTG GGG GCG GCG TGA 3ʹ′
5ʹ′ CAC CCC CGC CGC ACT AGA TCT CAT 3ʹ′ R1218XbaLS
XbaI

Amino acid sequence
MNVNVVGGISQWDTVQADAWWSAAVGPATNGEISMSAHGRPIHLGGFLIA 50
GNVTHSHPSWRHPRSDPGFLTPEYYQHLGRVFERAKLDFVFFADNSATPA 100
SYRNDIRDPLARGTQSAAGLDPRFVVPVVAGVTRNLGIVSTTSATFYSPY
150
DLARSFATLDHLTHGRVGWNVVTSNTTVEAQNFGLARHLDHDVRYDRAEE 200
LLEVAFRLWASWDDGALIQDKEAGVFADPDLIHRLDHHGENFDVRGPLSV 250
PRSPQGRPVIFQAGSSTRGRDFAARWAEAIFEIDPTSVGRKAYYDDIKSR
300
ASDFGRDPDGVKILPSFIPFVGETESIAREKQAFHNELADPTDGLITLSV
350
HTDHDFSGYDLDAVIADIDVPGTKGLFEVARSLSVNENLTLRDIGKLYAQ
400
GVLLPQFVGTAAQVADQIEAAVDGGEADGFLFSAGYTPGGFEEFADLVIP
450
ELQRRGRFRTEYTGSTLREHLGLPADANLVPVPRKAVGAA
34

6.2 Appendix B
YEME broth (100 mL)
0.3 g Difco Yeast extract
0.5 g Difco Pepto (bacto)
0.3 g Malt extract
1.0 g Glucose
34.0 g Sucrose
2 mL/L2.5 M MgCl2·6 H2O

6.3 Appendix C
pBluescript

(Addgene)

35

6.4 Appendix D
pMAL-c2

(Addgene)

6.5

Appendix E
Before transforming the ISGA 1218 gel purified fragment into the EcoRV linearized

pBluescript vector, the determination of the amount of ISGA 1218 required to achieve a 3:1
vector to insert ratio was required.
(1473 bp ISGA2 1218) / (3000 bp pBS) = 0.479 ng
0.479 ng * 3 = 1.437 ng ISGA 1218 / 1ng pBS * 100
= 147.3 ng ISGA 1218 required
36

Due to the poor recovery of the ISGA 1218 gel purified fragment, there was an
insufficient amount of DNA to prepare a 3:1 vector to insert ratio. Rather, a 1:1 ratio was
used.
(1473 bp ISGA2 1218) / (3000 bp pBS) = 0.479 ng
0.479 ng ISGA 1218 / 1ng pBS * 100
= 47.9 ng ISGA 1218 required

All calculations regarding the transformation efficiency of pBS were performed using
the number of blue colonies present on each control reaction. On each control plate, white
colonies were present surrounding the blue colonies due the diffusion of β-lactamases
expressed from the ampR gene within pBS allowing for the growth of competent cells not
containing pBS, and the associated ampR gene.

As such, the white colonies were not

included. To determine the fraction of pBS that could be transformed into each competent
cell, the number of transformants in the 10 ng uncut pBS control was used. In the sample
there was 10 ng of pBS in 25 µL ligation reaction. From this reaction volume, 2.5 µL (1 ng
pBS) was transferred into 1 mL of liquid broth, of which 100 µL (0.1 ng) was spread onto
ampicillin nutrient agar plates. To determine the number of molecules of pBS transferred,
the weight of one molecule of pBS was determined to be 3 x 10-18 g/molecule.
pBluescript = 3000 bp
One base pair = 660 g/mol
3000 bp * 660 g/mol = 2 x 106 g/molecule
2 x 106 g/molecule / 6 x 1023 g/molecule = 3 x 10-18 g/molecule

37

Using the weight of one molecule of pBS, I was able to determine the number of
molecules transferred in the 100 µL (0.1 ng) volume to be 3 x 107 molecules. From the
number of blue colonies visualized on the 10 ng uncut pBS control, it was determined 1.98 x
10-5 molecules of pBS entered the cell.
1 x 10-10 g / 3 x 10-18 g/molecule = 3 x 107 molecules

The transformation efficiency of the DH5α cells was also calculated using the 10 ng
uncut pBS control to be 5940 colonies/ng.
594 colonies / 0.1 ng pBluescript = 5940 colonies/ng

The effects of the ligation reaction on transformation efficiency was determined by
using the 10 ng uncut pBS with the complete ligation reaction. To determine this value, the
same calculation was used as for the transformation efficiency, using the average counted
colonies from the 10 ng uncut pBS (ligation reaction) transformation.

In the 100 µL

transformation reaction, 316 colonies were counted, giving a transformation efficiency of
3160 colonies/ng. This data suggests that the addition of the ligation reaction does decrease
transformation efficiency. The percent of decrease in efficiency was calculated.
(5940 – 3160) / 5940 = 0.468 * 100 = 47 %

In the 100 ng linearized pBS (no ligation reaction) control, the transformation
efficiency theoretically should have been observed as 0, as without DNA ligase, the plasmid
38

would be unable to recircularize and would be transformed into E. coli DH5α as a linear
plasmid, to be immediately degraded within the cell. However, 192 blue colonies were
observed on this control, suggesting an incomplete EcoRV restriction digest of pBS prior to
the ligation reaction. As 100 ng linearized pBS was added to a 25 µL, when 2.5 µL of the
ligation reaction was transferred into 1 mL of liquid broth, 10 ng of linearized pBS was
transferred. Of which, 1 ng of pBS was plated onto ampicillin containing nutrient agar,
resulting in 192 colonies/ng.

The efficiency of the digestion was calculated using the

transformation efficiency of pBS.
(192 colonies/ng) / (5940 colonies/ng) = 0.032 * 100 = 3.2 %

Using this same calculation, the percent recircularized in the 100 ng linearized pBS
(ligation reaction) control could be determined.
(464 colonies/ ng) / (5940 colonies/ng) = 0.078 * 100 = 7.8 % recircularized

The blue to white colony ratio for the 100 µL ISGA 1218 + pBS transformation was
68:1. To improve this ratio, a 3:1 vector to insert ratio should have been used. Due to our
poor QiaxII recovery of the ISGA 1218 fragment, the transformations were prepared using a
1:1 vector to insert ratio.

39

Table 10 Transformation efficiency of control reactions and ISGA 1218 + pBS
experimental reaction.
Transformation DNA form Blue (# of colonies) White (# of colonies
10 ng pBS
ccc
594
(- ligation reaction)
10 ng pBS
ccc
316
(+ ligation reaction)
100 ng pBS
linear
192
(- ligase)
100 ng pBS
linear
464
(+ ligase)
ISGA 1218 + pBS
linear
683
5

40