The unsuccessful attempt to express ldhA from yogurt: part 1

Abstract
Lactate dehydrogenase A is a gene that provides the instructions which encode for a protein also
known as Lactose dehydrogenase A. The protein itself catalyses the conversion of Lactate and
NAD to pyruvate and NADH in step ten of anaerobic glycolysis. Designing primers with
restriction sites plasmid pET28b was inserted into gene ldhA. Upon insertion it was then
transformed using E. coli to produce colonies.
Introduction
Yogurt has become widely known for its probiotic contents. The 1992 Codex Alimentarius of
1992 defines yogurt as a coagulated milk product that results from the fermentation of lactic acid
in milk. Fermentation is always performed by Lactobacillus bulgaricus and Streptococcus
thermophilus. (Adolfsson et al. 2004). L. bulgaricus is a representative of a heterogeneous group
of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt
production. (van de Guchte M et al, 2006). L. bulgaricus plays its role in yogurt production
mainly by producing l-lactate dehydrogenase. L-lactate dehydrogenase is a key enzyme that
couples l-lactate production to reoxidation of NADH formed during glycolysis. (Toyoda K et al,
2009). ldhA overseas the conversion of Lactate and NAD to pyruvate and NADH in step ten of
anaerobic glycolysis. Within the human body, ldhA is predominantly found in skeletal muscle
and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH.
However, LDH-A is overexpressed in many tumours and has therefore emerged as an attractive
target for anticancer drug discovery (Dempster S et al, 2014). Further studies in to ldhA need to
be performed in order to fully understand its nature so that he can be inhibited in the case of
tumors. In this study we aimed to investigate the gene ldhA from the bacteria Lactobacillus
bulgaricus from a sample of Chobani Greek yogurt. We attempted to see how the sequence of
this gene differs in bacteria isolated from commercially available yogurt, compared to the
standard published sequence.
Materials and Methods
Primer design
Using the software ApE, DNA sequences of LDHA were analyzed and primers were designed.
The PCR primer had 2 restriction enzyme recognition sites NheI and XhoI that enabled the
product to be cut by the desired restriction enzyme and ligated.
Cultures and purification
Lactobacillus bulgaricus collected from the yogurt sample, Chobani, it was grown in LB broth.
The harvested DNA was then quantified and purified via instructions from DNeasy 96 blood &
tissue kit for purification of total DNA from various sources. PCR was performed to amplify
ldhA
Figure 1 shows the reagents used to prepare for PCR

Figure 1: PCR reagants concenrtations

Gel electrophoresis
Gel electrophoresis was performed after almost every step so that we could observe if there was
anything present. Figure 2 shows where Chobani had no bands indication g the presence of ldhA.
Figure 2: gel electrophoresis results with only ladder bands present showing no signs of ldhA in
initial Chobani sample
Column Purification
Following instructions from the QIAquick Spin handbook, column purification was performed
on a different sample from CW.
Ligation/Transformation
Ligation was preformed to insert ldhA into the plasmid pET28b using their complementary
sticky ends from the resection enzyme digest (primers). Activia used from here on out.
Figure 3 shows the concentrations. Our product was then plated on purified through boiling and
plated on LB kanamycin plates. Figure 4 shows the result.
Figure 3: Ligation reagent concentrations.

Figure 4: LB cultures. The empty one is from the Activia sample and the one full of colonies is
from JB
Results
Initial DNA concentration from Chobani was 23.4 ng/ul
The gel electrophoresis in figure 1 showed no signs of DNA. Figure 2 shows the result of the
colony growth attempt on Lb + kan.
DNA conc form Activia’s sample was 52.ng/ul
Discussion
We hit a wall with Chobani immediately after culturing. The DNA concentration was 23.4 ng/ul.
It was underwhelming considering the preferred concentration is 25ng/ul. Our sample missed the
mark by 1.6. Figure 2 confirmed that ldhA was not successfully isolated. From then on we did
not have our own samples. We were assigned CW’s brown cow sample. The column
purifications were all performed with that sample. It was determined that the lack of success with
Chobani was not by personal error but it was the yogurt itself since all the Chobani samples
failed to perform. At ligation we were introduced to activia’s sample which had a DNA
concentration of 52.ng/ul which has so far been successful. Attempts at ligation were
unsuccessful because there was no growth on both + kan and -kan plates. Figure 4 shows the
plate from my activia samples and one from JB.

Citations

van de Guchte M, Penaud S, Grimaldi C, et al. The complete genome sequence of Lactobacillus
bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci U S A.
2006;103(24):9274-9279. doi:10.1073/pnas.0603024103

Adolfsson O, Meydani S, Russell R. Yogurt and gut function. Am J Clin Nutr.  2004;80:245–
256. 

Toyoda K, Teramoto H, Inui M, Yukawa H. The ldhA gene, encoding fermentative L-lactate
dehydrogenase of Corynebacterium glutamicum, is under the control of positive feedback
regulation mediated by LldR. J Bacteriol. 2009;191(13):4251-4258. doi:10.1128/JB.00303-09

Dempster S, Harper S, Moses JE, Dreveny I. Structural characterization of the apo form and
NADH binary complex of human lactate dehydrogenase. Acta Crystallogr D Biol Crystallogr.
2014;70(Pt 5):1484-1490. doi:10.1107/S1399004714005422
 The unsuccessful attempt to express ldhA from yogurt: part 2

Abstract
Lactate dehydrogenase A is a gene that provides the instructions which encode for a protein also
known as Lactose dehydrogenase A. The protein itself catalyses the conversion of Lactate and
NAD to pyruvate and NADH in step ten of anaerobic glycolysis. Designing primers with
restriction sites plasmid pET28b was inserted into gene ldhA. Upon insertion it was then
transformed using E. coli to produce colonies which we had hoped would’ve expressed ldhA.
SDS PAGE and a Bradford assay all support that this experiment was unsuccessful

Introduction
Yogurt has become widely known for its probiotic contents. The 1992 Codex Alimentarius of
1992 defines yogurt as a coagulated milk product that results from the fermentation of lactic acid
in milk. Fermentation is always performed by Lactobacillus bulgaricus and Streptococcus
thermophilus. (Adolfsson et al. 2004). L. bulgaricus is a representative of a heterogeneous group
of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt
production. (van de Guchte M et al, 2006). L. bulgaricus plays its role in yogurt production
mainly by producing l-lactate dehydrogenase. L-lactate dehydrogenase is a key enzyme that
couples l-lactate production to reoxidation of NADH formed during glycolysis. (Toyoda K et al,
2009). ldhA overseas the conversion of Lactate and NAD to pyruvate and NADH in step ten of
anaerobic glycolysis. Within the human body, ldhA is predominantly found in skeletal muscle
and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH.
However, LDH-A is overexpressed in many tumours and has therefore emerged as an attractive
target for anticancer drug discovery (Dempster S et al, 2014). Further studies in to ldhA need to
be performed in order to fully understand its nature so that he can be inhibited in the case of
tumors. In this study we aimed to investigate the gene ldhA from the bacteria Lactobacillus
bulgaricus from a sample of Chobani Greek yogurt. We attempted to see how the sequence of
this gene differs in bacteria isolated from commercially available yogurt, compared to the
standard published sequence.
Materials and Methods
Gene identification – Histidine tag
Ecole I was used to produce ldhA. It was then lyses (break open). Histidine tag was then
introduced. The sample was then purified via gravity flow purification.
SDS PAGE
30 micrometers of the samples were mixed with water and buffer before being loaded into
individual wells. SDS was ran at 200V. Figure 1

Figure 1: SDS PAGE result with no bands. From left to right in the wells they’re the DNA
ladder, pellet, flowthrough, wash and Elution 1-4

Bradford Assay  
A standard curve was created using albumin. Using a one-to-one ratio of each fraction mixed
with buffer and the Bradford Reagan. The absorbance of all samples was measured at 595 nm
using a spectrophotometer at 595 nm. Figure 2 
Figure 2: Standard curve for Bradford. Due to human error a few of the concentrations were
zero. In attempt to rectify this they were dropped for the sake of the slope. This Bradford showed
that there was a low concentration of protein in our sample

Protein Assay
A standard curve was created using albumin. Using a one-to-one ratio of each fraction mixed
with buffer and the dye Reagan. The absorbance of all samples was measured at 595 nm using a
spectrophotometer at 595 nm. 
Enzyme Assay
An attempt to plot a slope utilizing NADH levels within sample every 15 seconds for 150
seconds.
Results
Figure 1 shows that there was no protein present in samples,
The Bradford macro assay you’ll do that the protein concentration was too small to be detected.
The Bradford micro assay returned figure 2.
The enzyme assay returned a slope of zero
Discussion
This experiment was a complete unsuccessful. It was to be expected when we started switching
between samples within the room. Initially we were using activia but that also yielded no results.
For us while other groups were going strong. From the beginning to end of this experiment we
went from CW’s sample for the column then activia’s was successfully harvested. Activia passed
a few steps but then it failed at E.coli growth of pET28B. By the time we got to the titration it is
believed that we were using a sample from RR. It hasn't been checked the other nano spec hence
its DNA concentration was unknown. While other group’s standard Bradford assay yielded
viable absorbance, ours only yielded absorbance for three fields. The other concentrations
yielded zeros and that hindered the production of a slope, so they were left out of the table. That
leads to some skepticism around the data from that slope equation. The bio rad assay was more
successful and showed that we yielded a low amount of protein in are collected samples.
Ultimately did not come as a surprise when the Western blot and SDS page in figure 3 concluded
that we had nothing present in our sample. The lack of success from this experiment seems to
have started at LB plating on E. coli. It would appear that the plasmid did not make it inside E.
coli with the initial sample because pETB28 is kanamycin resistant in the original sample. The
SDS page, protein assay, and ends and assay all agreed that there was no LDHA present in our
sample. The bioinformatics Blast further asserted that they’re being only 3 matching based pairs
with the plasmid. In conclusion this experiment was unsuccessful
 Citations

van de Guchte M, Penaud S, Grimaldi C, et al. The complete genome sequence of Lactobacillus
bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci U S A.
2006;103(24):9274-9279. doi:10.1073/pnas.0603024103

Adolfsson O, Meydani S, Russell R. Yogurt and gut function. Am J Clin Nutr.  2004;80:245–
256. 

Toyoda K, Teramoto H, Inui M, Yukawa H. The ldhA gene, encoding fermentative L-lactate
dehydrogenase of Corynebacterium glutamicum, is under the control of positive feedback
regulation mediated by LldR. J Bacteriol. 2009;191(13):4251-4258. doi:10.1128/JB.00303-09

Dempster S, Harper S, Moses JE, Dreveny I. Structural characterization of the apo form and
NADH binary complex of human lactate dehydrogenase. Acta Crystallogr D Biol Crystallogr.
2014;70(Pt 5):1484-1490. doi:10.1107/S1399004714005422