Identification of pBLU with transformation, purification, and electrophoresis using
E.coli and plasmid #10
Written By:
Walker N. Foland
LBS 145
Section Monday 7-9:50pm
Rick Chalmers and Dan Gutteridge
April 27, 2004

Abstract: by Walker Foland
The goal of the study was to investigate the identity of an unknown plasmid. To do that, three experiments were performed. The first experiment of the study was transformation. Transformation can be best defined as the uptake of plasmid DNA into a bacterium. This method was used to allow the Escherichia coli to display antibiotic resistance characteristics brought on by the gene expression of the plasmid. Three different antibiotics were used to isolate the transformed bacteria; Ampicillin,
Tetracyclin, and Kanamycin. The second portion of the study was amplification and purification. These processes together isolated the unknown plasmid and amplified its quantity in order for further analysis to occur. The third and final portion of the study was
DNA fingerprinting. It consisted of two parts; restriction enzyme digest and electrophoresis. In the restriction enzyme digest the plasmid was cut at specific base pair locations with two enzymes. The lysed DNA were ran in an electrophoresis gel. The unknown plasmid was found to be Ampicillin resistant in both amplification and transformation. Using a Hind III and Ava II restriction digest, bands of DNA were observed through gel electrophoresis. Those bands were compared to a known DNA restriction digest known as Lambda HindIII digest. Using a logarithmic graph, the calculated base pair lengths corresponded to the predicted base pair lengths of pBLU.
Then a second electrophoresis gel was ran with the known plasmid pBLU along with the unknown plasmid. The unknown bands and pBLU bands matched perfectly. It was concluded that the unknown plasmid DNA was pBLU.

Introduction: by Walker Foland
Bacteria are among one of the oldest life forms on Earth. It exsisted long before humans or any other type of multicellular organism (Wassenaar 2003). Not only are bacteria one of the oldest life forms on the planet, it is also one of the most parasitic.
Many diseases have risen due to bacterial infection in humans and all creatures alike.
Such a disease, which can be caused by bacterial infection, is Pneumonia.
Pneumonia is an infection of the lung tissue, which results in difficult breathing, possible delusions, fever, shivering, coughing, and chest pain (Net Doctor 2003). It is caused by a bacterium called streptococcus pneumoniae. This disease is seen as being quite dangerous to the elderly. It has much more of a hold on those with weakened immune systems resulting in a much higher rate of death. Overall bacterial infections have been a bain to the human race. Pneumonia is currently the 7 th
leading cause of death in the US.
In 2001 it killed 62,034 people in the United States alone (CDC 2004). Bacteria has been studied for hundreds years, but has not been thoroughly understood or analyzed until the past 100 years. The development of antibiotics was and is still a weapon of massive importance to the prevention of disease. However, bacteria have many methods to create resistance to adapt to the onslaught of microscopic assassins thrown at them.
Bacteria have many dynamic characteristics, which enable them to change. One of those characteristics, which allow them to adapt so quickly and successfully, is a circular piece of DNA known as a plasmid (Krha et al. 2003). Plasmid DNA can be exchanged from cell to cell in the bacterial world. Plasmids carry instructions for the bacterial cell to develop proteins to allow a certain resistance to any number of diverse

conditions (Krha et al. 2003). Understanding how plasmids work can significantly impact cures for bacterial infection and therefore, lower cases of disease caused by them.
Escherichia coli was used in this study to express the gene coding of the unknown plasmid. To display such characteristics, the bacteria had to uptake the plasmid. The process of enabling bacteria to uptake a plasmid is known as transformation.
Transformation has duel importance. One, only a small amount of DNA was given to analyze, to go on with the planned experiments more would be needed. Two, in order to test the plasmid for antibiotic resistance it must have a host in order to translate resistant proteins. LB agarose plates were treated with three different antibiotics in order to grow the transformed bacteria. The prediction was that the successfully transformed bacteria would survive on one or more of the antibiotically treated plates. This would isolate transformed bacteria only. Once antibiotic resistant bacteria were isolated successfully, then amplification in Luria-Bertani (LB) broth (also treated with antibiotics) occurred.
Amplification created a large colony of bacteria and therefore synthesized more plasmid
DNA. Using three different chemical solutions the transformed and amplified bacteria were broken down to isolate the DNA plasmid only. Then through restriction digest the unknown plasmid was lysed at specific base pair locations to allow DNA fingerprinting.
This would enable the calculation of fragment weight for the plasmid through a line equation from graphing a Lambda Hind III ladder digest. Through the processes of transformation, amplification, purification, and DNA fingerprinting positive identification of the unknown plasmid is possible.

Methods: by Walker Foland
An unknown plasmid was investigated using Escherichia coli. The first of three steps in identifying the unknown plasmid was a process called transformation as described on page 105 of the LBS 145 lab manual. The LBS 145 lab provided all materials for all of the following procedures. The amount of E.coli used in the transformation procedures were cut in half from 50 ml to 25 ml. Therefore the amount of CaCl
2
used in the procedures were also cut in half. Two controls were used for spreading the E.coli onto the
LB agarose plates. Both controls contained no antibiotic. One was spread with transformed E.coli and was labeled con +, indicating the presence of transformed bacteria. The other was not spread with transformed bacteria and was labeled con- to indicate the absence of transformed E.coli. Three antibiotics were used in the process of pouring the LB agarose plates prior to transformation. The process of pouring agarose plates is described on page 130 of the LBS 145 lab manual. Appropriate amounts of antibiotic were applied to certain plates. In the experiment there were eight plates in all.
Each labeled appropriately con+, con-, amp+, amp-, tet+, tet-, kan+, and kan-. Kan, amp, and tet stand for the antibiotics, Kanamycin, Ampicillin, and Tetracyclin respectively.
The plates were all incubated at 37 ˚C for 18 hours.
The second of the three experiments in identification of the unknown plasmid was amplification and purification. The processes of both are explained on pages 99 through
102 of the LBS 145 lab manual. Five centrifuge tubes were used in amplification. Each one contained 20 μl of antibiotic and 2ml of LB broth. Three tubes had Ampicillin antibiotic and the remaining two had Tetracycline and Kanamycin. After amplification, purification began. Purification was performed with 3 trials instead of one described in

the lab book procedures. Once purification was complete a new microfuge tube was used to mix 6 μl of DNA with 2 μl of loading dye. Then the mixture was centrifuged and pipetted into an electrophoresis gel that was prepared by the LBS 145 lab TA’s. The
DNA remaining was stored at –20 ˚C.
The third and final portion in the process of identifying the unknown plasmid was
DNA fingerprinting. Prior to gel electrophoresis, a restriction enzyme digest was performed on the DNA subject to analysis. Two trials were performed for the first restriction digest corresponding to two lanes in gel one. A 1.5XKGB buffer concentration presented ideal efficiency for digestion; therefore, both microfuge tubes were filled with 5.63 μl of 4X KGB buffer, 7.4 μl of DNA, and 1 μl of Hind III and Ava
II restriction enzymes. Those amounts were calculated with the equation V
1
C
1
=V
2
C
2 creating a final volume of 15 μl
.
The procedures for restriction enzyme digest are described on page 111 of the LBS 145 lab manual. The microfuge tube was centrifuged for 5 seconds at 12,000 RPM and then incubated in a rotating water bath at 37 ˚C overnight. A digest time of one hour was required for complete digestion, however overnight digestion was performed for extra assuredness. Before electrophoresis could begin, the gel had to be prepared. An Erlenmeyer flask was filled with 100ml of electrophoresis buffer and .8 grams of agarose powder. The mixture was micro waved for approximately 2.5 minutes. Once the mixture cooled to 60 ˚C, 4 μl of EtBr was added and mixed. The procedures for pouring the agarose electrophoresis gel are on page 119 in the
LBS 145 lab manual. The DNA was mixed with a 2 μl of loading dye according to the digest volume. The DNA was loaded into the gel using a pipette. The gel was hooked up to electrical leads and a voltage of 80-90 volts was applied. A second gel was prepared

with a second digest and all procedures were the same except a known DNA plasmid was used along with the unknown plasmid.
Results: by Walker Foland
Transformation was performed four different times (Figure 1). On the fourth attempt successful transformation was achieved. The first set of plates had only growth on the LB Agar control plates with no growth on the antibiotic treated plates. The second set of plates had similar results. The third set of plates had enormous growth on the AMP
+ , AMP – , and control plates. More growth was observed on the AMP +/- plates than on the control plates.
After the fourth attempt at transformation E.coli growth was observed on both controls and the Ampicillin treated plate (Figure 2). One control plate (LB+) containing
LB Agar and transformed E.coli cells had thick growth with many colonies. The other control plate (LB-) contained LB Agar and non-transformed E.coli cells. Growth was observed to be thick and abundant, nearly exactly the same as the LB+ control. The growth observed on the LB+ Ampicillin plate was spotted and scarce compared to the two control plates.
Amplification and purification of the plasmid DNA from the E.coli brought forth strong results. In amplification, bacterial growth was observed in all three of the LB/
Ampicillin centrifuge test tubes (Figure 3). No growth was observed in the Tetracycline and Kanamycin control tubes. Many more steps toward purification finally lead to the gel electrophoresis of the purified plasmids. DNA was found to be present in the gel test for all three trials performed for purification (Figure 4).

In the enzyme restriction digest two restriction enzymes were used for the plasmid
DNA, Ava II and Hind III (Figure 5). The buffer used for that digest was 4XKGB buffer, which was diluted to 1.5XKGB (Table 1). Lambda DNA was digested and cut to make fragment sizes of 2027, 2322, 9421, 564, 6683, and 27493 (Table 2).
Electrophoresis was performed twice with the digested DNA plasmid. The first gel ran had one control, a Lambda DNA Hind III digest. This revealed an obvious DNA ladder for comparison against all other plasmid digests (Figure 6). Using a logarithmic graph, general DNA molecular weight could be calculated from the Lambda Hind III digest (Figure 7).
The first gel revealed the near positive identification of pBLU (Figure
8). Expected visible digestion fragment sizes (in base pairs) of pBLU with Hind III and
Ava II digest were 1398, 1591, and 2226. The calculated fragment sizes, from the
Lambda Hind III digest, for pBLU were 2521, 1616, and, 1375 base pairs in length
(Table 3) .
In the second gel, pBLU was used as a known plasmid along with the unknown plasmid, suspected to be pBLU. The known and unknown digests were shown to be identical (Figure 9). This confirmed that pBLU was the unknown plasmid.

Discussion: by Walker Foland
The objective of this study was to be able to positively identify an unknown plasmid. There were three large portions to the overall identification of the unknown plasmid DNA. The first portion of the study was called transformation, which consisted of two parts. The process of transformation involves taking bacterial cells and using a series of chemicals and procedures to enable a DNA plasmid to be diffused. This allows the bacterial host to translate proteins from the plasmid to acquire antibiotic resistance and other characteristics. The second portion was called amplification and purification, it consisted of two parts as well. The process of amplification involves growing transformed bacteria in a bath of LB broth and antibiotic in order to increase the amount of DNA being studied and to further support the presumed antibiotic resistance. The third portion of the study was called DNA fingerprinting, it was made up of three parts. The process of DNA fingerprinting involves using restriction enzymes to cut a plasmid at specific locations so it can be differentiated from other plasmids and DNA under the same digest in an electrophoresis gel. The hypothesis of this study was that positive identification of the unknown plasmid DNA could be achieved through these three portions of the study. A large amount of evidence was gathered and all them point to the unknown plasmid being pBLU.
The first step toward transformation was pouring LB agar plates. Eight plates were poured and all but two control plates were treated with calculated amounts of antibiotic. Three different antibiotics were used: Tetracyclin (tet), Kanamycin (kan), and

Ampicillin (amp). The second step in transformation was preparing the E.coli bacteria.
Twenty-five ml of bacteria were used for transformation. After transformation of the
E.coli was complete, the bacteria had to be spread over all of the LB plates. Two types of bacteria were used for spreading on the plates. One type of bacteria was transformed and the other type of bacteria were not transformed with the unknown DNA plasmid. The plates with transformed DNA on them were labeled with a (+) along side there corresponding antibiotic. Those with untransformed DNA were labeled with a (-) along side there corresponding antibiotic. All of the plates were incubated at 37 ˚C for 18 hours.
Growth was observed on the amp+ plate and on both controls. This supports the claim that the unknown plasmid gives the E.coli bacteria Ampicillin resistance and that transformation was done successfully.
Transformation was performed four times before it was successful. In the first two times no growth was observed on anything but the control plates. This was most likely due to unsuccessful transformation of the plasmid across the bacterial membrane. The third attempt had enormous growth on both amp+ and amp– plates, along with the controls. This was most likely due to contamination of the amp plates.
The second portion of the study to identify the plasmid is known as amplification and purification. Amplification was performed with five centrifuge tubes all containing
LB broth. Three tubes contained Ampicillin and the remaining two contained Tetracyclin and Kanamycin. All tubes had transformed E.coli put in them from the bacterial growth on the amp+ plate. After an overnight incubation in a gyrating oven set at 37 ˚C, growth was observed in the Ampicillin treated tubes, but no growth was observed in the

Tetracylcin or Kanamycin tubes. This further supports the claim that the unknown plasmid gives the E.coli resistance to Ampicillin.
Purification was performed in order to isolate the plasmid from the E.coli. After breaking apart the cell membrane and precipitating the plasmid separate from the other types of DNA from the bacteria, 6 μl of plasmid DNA were mixed with 2 μl of loading dye in a separate microfuge tube. This mixture was centrifuged and ran in a gel in order to observe the presence of plasmid DNA. Three trials were performed and all three trials showed strong bands of plasmid DNA indicating successful purification. The DNA remaining was put in a freezer at –20 ˚C.
The third and most important portion of the study was DNA fingerprinting. This consisted of two parts. The first part was the restriction enzyme digest. The restriction enzyme digest used on the plasmid had two enzymes, Hind III and Ava II. This was a good combination of enzymes because it cut all of the possible plasmids at different locations making for different DNA fragment lengths. The formula V
1
C
1
=V
2
C
2 was used in order to calculate the correct amounts of DNA, 4xKGB buffer, and restriction enzymes to put into a new microfuge tube for digestion. Two trials or microfuge tubes were used in the digestion, each tube identical to the other. Once all needed chemicals were in the microfuge tubes, they were both incubated overnight to achieve complete digestion, however only one hour was really necessary.
The second part of DNA fingerprinting was running the gel electrophoresis. Two gels were run. The first gel was known as gel 1 and the second was known as gel 2. The agarose electrophoresis gel was put into a bath of electrophoresis buffer. Just enough buffer was added onto the gel to barely cover its surface by 1mm. The plasmid DNA that

had been digested was mixed with 2-3 μl of loading buffer and centrifuged. The DNA was then pipetted into the electrophoresis gel along with a prepared Lambda Hind III digest. The gel was run for 1 hour and 15 minutes with a voltage of 80-90 volts. The
Lambda ladder digest had known values for the bands of DNA, which enabled a log graph to be made. The log graph was used to calculate the base pair lengths of the unknown plasmid. The calculated base pair lengths of the plasmid were 2521, 1616, and,
1375. This data matched up very well with the predicted base pair lengths of pBLU which were 2226, 1591, and 1398.
The first gel 1 performed was actually unsuccessful. Appropriate measurements and calculations were performed, yet the data came out to match closest to pAMP. This was most likely due to innaccurate measurements from a blown up polaroid of the gel. It was also likely due to imperfections and impurities in the agarose gel combined with a premature gel run time. The second gel was much more accurate due to a longer running time. Measurements were also much more accurately calculated using computer software.
Measurements of the gel bands were calculated using pixels and then converted to mm.
Gel 2 was performed to confirm the suspition of the unknown plasmid being pBLU. Gel 2 was ran with one unknown, and two knowns. One known was the Lambda ladder, and the other was pBLU. All other procedures were identical to the first gel. The second gel revealed identical bands for the unknown and pBLU. The hypothesis was accepted and confirmed the prior data that the unknown plasmid was in fact pBLU.

References: by Walker Foland
American Lung Association. 2004. Pneumonia.
http://www.lungusa.org/site/pp.asp?c=dvLUK9O0E&b=35691.
Accessed 4-18-04.
CDC. 2004. Deaths/Mortality. http://www.cdc.gov/nchs/fastats/deaths.htm.
Accessed 4-21-04
Krha, Maleszewski, Wilterding, Sayed, and Luckie. 2004. LBS-145 Cell and Molecular
Biology Course Packet. 48-68
Net Doctor. 2003.Pneumonia. http://www.netdoctor.co.uk/diseases/facts/pneumonia.htm.
Accessed 4-18-04.
Wassenaar, T.M. 2003. Special feature: Bacterial Diseases in History.
http://www.bacteriamuseum.org/niches/features/diseasehistory.shtml.
Accessed 4-18-04.

written by Walker Foland
All four attempts Fourth and successful attempt
First Attempt Second Attempt Third Attempt
Figure 1: Four attempts at Transformation.
These are all of the plates that went into attempting transformation. In the first and second attempts no growth was found on anything but the control plates. The third attempt had enormous growth on the amp+/- plates and the con+/- plates.

Figure 2: LB Plates.
This is a picture of the LB Broth plates used in transformation.
Growth was only found on the Amp+ and control plates. The transformed E.coli cells were therefore resistant to the antibiotic Ampicillin. Growth on the control plates were so ubiquitous that it showed up on the plates as a thick film, not easily visible in this photograph.

Kanamycin Ampicillin Tetracycline
Figure 3: Amplification Centrifuge Tubes. (Top) These are pictures of the transformed bacterial amplification tubes for the amplification portion of the study. Each was treated with a different antibiotic (indicated below). Growth can be seen by the white pellet in the base of the centrifuge tube and the cloudy color of the fluid. Growth was found in the
Ampicillin tube, but not in the Kanamycin or Tetracycline tubes. (Bottom) All trials of amplification are seen here; Three trials of amp treated tubes and one trial of tet and kan treated tubes.

Figure 4: Presence of DNA.
This is a photograph of the miniprep gel in lab 2. This shows evidence of DNA after purification from transformed E.Coli bacteria. The bright colors displayed are from the traveling bands of DNA gradually collecting Ethidium
Bromide (EtBr) as it travels from cathode to anode on the agarose gel. There are three lanes due to three repetitions of the experiment to improve the probability DNA presence.

Figure 5: This is the plasmid map of pBLU, the predicted unknown plasmid yet to be identified. The remainder of the possible plasmid maps are available in the appendix. Cut sites are shown on each map labeled appropriately with which restriction enzyme used.
Below is a key to find the remaining maps.
Plasmid map
PUC 19
PBR322 pAMP pKAN pGPS3
PACYC177
Lambda
Appendix Page Number
1
2
3
4
5
6
7

Figure 6: DNA Fingerprint.
This is an illustration of the expected DNA fingerprint from Gel Electrophoresis for all seven plasmids. The far right side of the graphic shows the length of the DNA strands post-lysis in the Lambda DNA-Hind III Digest. It is measured in Base Pairs (BP). This is to give a reference in the digest to other plasmids and their DNA strand lengths. Compare the pBLU drawing to the photographs of the actual gel, the resemblance is impeccable.

Hind III ladder digest
100000
10000
1000
100
10
1
0
Line Equation y = 5E+07x
-2.4207
Hind III BP
Power (Hind III BP)
20 40
60 80
Figure 7: Log graph of Lambda Hind III digest. This graph provides the basis for calculating the molecular weight (number of base pairs) for the unknown plasmid.
Plugging in the number of mm of distance the unknown plasmid traveled through the gel would give the approximate molecular weight of the unknown plasmid.

Figure 8: Identification of unknown plasmid DNA. This is a photograph of the first gel electrophoresis. On the far left there is two trials of the unknown plasmid and one trial of the Lambda Hind III digest. Comparing this photograph to that of the predicted gel graphic in figure 5, visually the unknown plasmid can be seen as being pBLU.

Figure 9: Positive Identification of unknown plasmid . In this photograph there are two lanes of DNA. One is the unknown plasmid and the other is the known plasmid. The known is pBLU and perfectly mirrors the unknown plasmid. To the far right of the photo there is a Lambda Hind III ladder.

Table 1: Restriction digest recipe . This is a table giving direction to the amounts of
DNA, Restriction Enzyme, Buffer, and dH2O that should go into digesting each one of the plasmids. It also gives calculated digest times for each.
DNA (µl ) Enzyme (µl ) Enzyme 2 (µl ) Buffer (µl ) DH2O Total Digest Time
PAMP- 7.375 µl Hind III- 1 µl Ava II- 1 µl N/A 15 µl 1:00
PUC 18/19- 7.375 µl
PBR322- 7.375 µl pBLU- 7.375 µl pKAN- 7.375 µl pGPS3- 7.375 µl
Hind III- 1 µl Ava II- 1 µl
Hind III- 1 µl Ava II- 1 µl
Hind III- 1 µl Ava II- 1 µl
Hind III- 1 µl Ava II- 1 µl
Hind III- 1 µl Ava II- 1 µl
PACYC177- 7.375 µl Hind III- 1 µl Ava II- 1 µl
4 X KGB-
5.625 µl
4 X KGB-
5.625 µl
4 X KGB-
5.625 µl
4 X KGB-
5.625 µl
4 X KGB-
5.625 µl
4 X KGB-
5.625 µl
4 X KGB-
5.625 µl
N/A
N/A
N/A
N/A
N/A
N/A
15 µl
15 µl
15 µl
15 µl
15 µl
15 µl
1:00
1:00
1:00
1:00
1:00
1:00

Table 2: Fragment sizes . This shows the entire set of fragment sizes acquired from the restriction enzyme digest of all of the plasmids. The two right columns describe the cut locations of plasmid by the restriction digest and which enzymes are doing the cutting.
The column labeled length is the base pair lengths of DNA resulting in the restriction digest.
Length (BP)
814
748
222
20
1264
950
661
42
2226
1591
1398
222
1390
1000
1744
770
664
303
279
249
222
89
3616
1798
888
222
1390
222
2506
510
434
222
155
42
1303
1215
766
435
222
27493
9421
6683
2322
2027
564
Order pAMP
1
2
3
4 pUC 19
1
2
3 pBR322 pBLU
1
2
3
4 pKAN
1
2
3
4
5
6 pGPS3 pACYC177
1
2
3
4
5
Lambda/ Hind III
1
2
3
4
5
6
4
5
6
7
8
1
2
3
5
6
7
8
9
1
2
3
4
From
Ava II- 2286
Ava II- 2951
Ava II- 1403
Ava II- 2796
Ava II- 97
Ava II- 403
Ava II- 2907
Ava II- 1706
Hind III- 2472
Ava II- 181
Hind III- 44149
Hind III- 27481
Hind III- 37466
Hind III- 25159
Hind III- 23132
Hind III- 36902
Ava II- 3161
Ava II- 1363
Hind III- 233
Ava II- 3161
Hind III- 447
AvaII-1837
AvaII-2059
Ava II- 1760
Hind III- 29
Hind III- 29
Ava II- 1136
Ava II- 1481
Ava II- 887
Ava II- 3504
Ava II- 779
Ava II- 1439
Ava II- 1599
Ava II- 1599
Hind III- 3190
Ava II- 4588
Ava II- 3038
Hind III- 234
Ava II- 1234
Ava II- 2068
Ava II- 2816
Ava II- 2048
Ava II- 139
Hind III- 3385
Ava II- 1625
To
Ava II- 2796
Hind III- 3385
Ava II- 1625
Ava II- 2951
Ava II- 139
Ava II- 1706
Ava II- 181
Hind III- 2472
Ava II- 2907
Ava II- 403
Hind III- 23132
Hind III- 36902
Hind III- 44149
Hind III- 27481
Hind III- 25159
Hind III- 37466
Hind III- 233
Ava II- 3161
Ava II- 1121
Ava II- 3383
AvaII-1837
AvaII-2059
Hind III- 447
Ava II- 3504
Ava II- 799
Ava II- 3726
Ava II- 1439
Ava II- 1760
Ava II- 1136
Ava II- 3726
Ava II- 887
Ava II- 1481
Ava II- 4810
Hind III- 3190
Ava II- 4588
Ava II- 4810
Hind III- 234
Ava II- 1234
Ava II- 2048
Ava II- 2816
Ava II- 3038
Ava II- 2068
Ava II- 1403
Ava II- 97
Ava II- 2286

Table 3: Calculated molecular weight of unknown plasmid . This table shows the distance traveled by the unknown plasmid fragments (left column) along with the corresponding molecular weight (center column). Molecular weight was calculated using the formula y = 5E+07x-2.4207 (right column). The calculations come very close to the predicated molecular weight of pBLU. BP stands for base pairs.
Band travel of
Unknown Plasmid (mm)
Calculated Predicted
Unknown Plasmid BP pBLU BP
59.6
71.62
76.56
2521.339824 2226
1616.175184 1591
1375.202158 1398

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