Essay: Successful Creation of Recombinant pET-41a(+)/EGFP Plasmid Confirmed by PCR and Restriction Digest

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Introduction

Plasmids are so helpful and important to understanding about genes in the field genetic. Fragment of DNA can ligate to plasmid vector to create a recombinant expression plasmid. In 1973, recombinant plasmid DNA was first achieved by Boyer and Cohen at Stanford University, California. In the beginning of this experiment, we will create a recombinant expression plasmid by ligation egfp cDNA insert into pET-41a(+) vector. Then, this recombinant expression plasmid will be transformed into E. coli cells for replication and cloning. After growth all culture of recombinant clones, they will isolate and purify through mini prep. Moreover, all recombinant plasmid will be determined and identify through restriction digest and PCR by run on gel electrophoresis. Finally, in the end of the experiment, we will using online tools to confirm the recombinant pET-41a(+)/egfp plasmid.

Method:

Ligation:

Egfp cDNA insert was ligated into pET-41a(+) vector to create a recombinant expression plasmid by setting up a total of five ligation reaction samples which were consisted of 20 ul volume. Ligation 1 had 1:1 molar ratio pET-41a(+) vector: egfp insert. Ligation 2 had 1:3 molar ratio pET-41a (+): egfp insert. They both consisted of NcoI/NotI cut pET-4a (+) DNA at a concentration of 25ng/ul, egfp insert DNA at a concentration of 7ng/ul, 10x ligase buffer, DNA ligase, and dH2O. Ligation 3 was known as a positive control, it consisted of uncut pET-41a(+)/EGFP recombinant plasmid DNA and sterile dH2O. Ligation 4 was known as a negative control which consisted of dH2O only. Ligation 5 was also known as a negative control which had everything as ligation 2, except DNA ligase control. These five ligation reaction samples were mixed gently in a microcentrifuge tube for about 5 seconds and incubated at room temperature. Secondly, all five ligations ran on 40ml of 0.8% agarose gel electrophoresis at 110V for about 45 minutes to visualize DNA.

Transformation:

The recombination expression plasmid will able to replicate and form multiple copies of itself during this transformation part of experiment by adding 20ul of the competent cell directly into 2 ul of four appropriate ligations. After incubated each tube on ice for five minutes, they were heated shock by put these in a 42C heat block for 2 minutes, then immediately removed and put these backs and incubated on ice for another 2 minutes. After that, Luria broth was added into each tube and incubator for 45 minutes at 37C. Then, 80ul from each tube were plated onto LB/Kan/IPTG plates, while 20ul of transformation 2 was added onto LB/Kan plate and 20 ul of transformation 4 was also added onto the LB only plate. Finally, all six plates were placed in the 37C incubator overnight to allow bacterial growth.

Miniprep:

After transformation, the cultures had been grown from a single colony which are picked from the transformations. A fluoresces green under UV light colony that was picked from an IPTG/Kan/LB plate is known as the recombinant plasmid. A white under UV light colony that was picked from an IPTG/Kan/LB plate also known as non-recombinant plasmid. A white colony that was picked from LB/Kan plate is known as unknown plasmid. The Promega Wizard miniprep kit was used to purify DNA from each of three cultures. However, to elute plasmid DNA from the minicolumn, double amount of Buffer EB was added into non-recombinant in accidently. Then, a spectrophotometer called a Nanodrop was performed to quantitate the concentration and the 260/280 ratio of three DNA plasmid.

Restriction Digest:

A total of nine restriction digest reactions were set up in this part of experiment. There were three single digests cut with Nco I enzyme, three double digests cut with Nco I and Not 1 enzyme, and three undigested for each recombinant, non-recombinant, and unknown plasmid DNA. All nine digestion tubes would have a total volume of 20 ul consisted of master mix, sterile water and our DNA plasmid. After microcentrifuge these nine tubes, they incubated in the 37C water bath for 1 hour. Finally, after finish track dye all nine digestion tubes, DNA ladder and all nine digestion tubes ran on 50ml of 0.9% agarose gel electrophoresis at 110V for about 45 minutes.
Gel Electrophoresis of PCR:

Unfortunately, we didn’t keep our DNA plasmid from previous Mini prep part of experiment, so we borrowed three DNA plasmids from our classmate group to do this PCR experiment. We used Primer 1 and Primer 2 which is specific for the egfp insert, and a region of the pET41a(+) vector, respectively in this PCR experiment. A recombinant, non-recombinant, unknown DNA plasmid that our classmate group isolated before, a positive control which contains pET/EGFP recombinant plasmid and a negative control which contains sterile water only were our five DNA templates in this PCR experiment. Firstly, we made a total of 20uL volume includes five PCR tube and master mix which consisted of 1X PCR buffer, 800 uM dNTPs, 0.5 uM Primer 1, 0.5 uM Primer 2, 2.5 units Taq polymerase and sterile water. Then, these five tubes were placed in thermocycler and incubated at 95C for 10 minutes. Next, they also incubate at 95 C for 1 minute, at 56 C for another 1 minute, and at 72 C for 1.5 minute for a total of 30 cycles. After that, they continued to be incubated at 72 C for 5 minutes for 1 cycle, stored them at 4 C to suspend reaction. Finally, after one week, track dye was added directly to all five tubes, then 10ul of DNA ladder and 6ul of each five tubes ran on 40 ml of 0.9% agarose gel electrophoresis.
Virtual Cloning:
The pEGFP-N1 plasmid nucleotide sequences were found in NCBI database. The WatCut restriction analysis tool and the AddGene Vector Database are two important online tool for analyzing information DNA. By using the WatCut restriction analysis tool, the restriction enzyme cut sites would be found. Also, using the AddGene Vector Database, we found the pET41a(+) vector sequence. After that, we generated the sequence of the EGFP/pET41 recombinant plasmid by combine all the pEGFP-N1 plasmid nucleotide sequences and the pET41a(+) vector sequence. Moreover, the location two sequences of the PCR primers and size of PCR product were determined in the sequence of the EGFP/pET41 recombinant plasmid by using WatCut online tool.
Results:
Ligation:
Key
1 DNA ladder
2-ligation #1 1:1 molar ratio pET-41a(+) vector: egfp insert
3-ligation #2 1:3 molar ratio pET-41a(+) vector: egfp insert
4-ligation #3 Circular recombinant pET41a(+)/EGFP plasmid
5-ligation #4 No DNA or DNA ligase control
6-ligation #5 No DNA ligase control
Figure 1: Gel electrophoresis of DNA ladder and five ligation reactions
Lane 1 shows the DNA ladder which contains a standard length of DNA fragments. Lane 2 contains 1:1 molar ration pET-41a(+) vector: egfp insert which shows a total of two bands. The top band presents the open circular form of plasmid DNA at 6kb and smear above the plasmid DNA. The bottom band presents the supercoiled DNA at about 0.74kb. Lane 3 contains 1:3 molar ratio pET-41a (+) vector: egfp insert which shows a total of two bands. The top band presents the open circular form of plasmid DNA at 6kb and smear above the plasmid DNA. The bottom band presents the supercoiled DNA at about 0.74kb. However, the bottom band in lane 2 looks more blur than compare to the bottom band in lane 3. Lane 4 contains a circular recombinant pET41a(+)/EGFP plasmid. It is known as positive control plasmid which shows a total of two bands at 10kb and 4kb. Lane 5 contains dH20 only which is also known as negative control, shows no band. Lane 6 contains 1:3 molar ratio pET-41a(+) vector: egfp insert without DNA ligase. It is known as a negative control plasmid which shows a total of two bands at 6kb and about 0.74kb.
Transformation:
LB/IPTG/ Kan (ligation #1) LB/IPTG/ Kan (ligation #2) LB/IPTG/ Kan
(ligation #3) LB/IPTG/ Kan
(ligation #4) LB/Kan (transformation#2) LB (transformation#4)
Green Colony 3 10 11 N/A N/A N/A
White Colony 1 8 N/A N/A 1 Lawn
Transformation efficiency 5.39112 Transformants/μg DNA
24.26
Transformants/μg DNA
17.52
Transformants/μg DNA
N/A 1.29
Transformants/μg DNA
N/A
Figure 2: Colony in Petri dishes and transformation efficiency
Figure 2 shows a total of green colonies which known as recombinant colony picked from LB/IPTG/Kan plate, white colonies which known as non-recombinant colony picked from LB/IPTG/Kan plate and white colonies which known as unknown colony picked LB/Kan plate in six Petri dishes. Plate 1 which consisted of 1:1 molar ratio pET-41a(+) vector: egfp insert from ligation #1 and LB/IPTG/Kan observed 3 green colonies and 1 white colony. Plate 2 which consisted of 1:3 molar ratio pET-41a (+) vector: egfp insert from ligation #2 and LB/IPTG/Kan observed 10 green colonies and 8 white colonies. Plate 3 which consisted of positive control from ligation #3 and LB/IPTG/Kan observed 11 green colonies, and no white colonies. There are no colony presented in plate 4 which consisted of negative control in ligation #4 and LB/IPTG/Kan. Plate 5 which consisted of a 1:3 molar ratio pET-41a (+) vector: egfp insert from ligation #2 and LB/Kan observed 1 white colony. Plate 6 which consisted of negative control from ligation #4 and LB only plate observed a solid “lawn” of bacteria. The transformation efficiency (TE) data were calculated by the number of transformants dived by the amount of plasmid DNA transformed in ug unit. We calculated 5.39112, 24.26, 17.52, 0, 1.29, 0 transformants/μg DNA in respectively plate #1, #2, #3, #4, #5 and #6.
Concentration(ng/ul) 260/280 ratio
Recombinant 47.3 1.93
Non-recombinant 28.5 2.01
Unknown 49.2 1.92
Miniprep:

Figure 3: The concentration and 260/280 ratio of plasmid DNA samples from our group data.
Concentration(ng/ul) 260/280 ratio
Recombinant 44.2 1.97
Non-recombinant 64.6 1.78
Unknown 58.0 1.76
Figure 4: The concentration and 260/280 ratio of plasmid DNA samples from our classmate data.
According to figure 3, the concentration of recombinant, non-recombinant, unknown is 47.3ng/ul, 28.5ng/ul, 49.2ng/ul respectively. They are quite too low compare to a typical concentration of plasmid DNA. From figure 3, there are 44.2ng/ul, 64.6ng/ul, and 58.0ng/ul from recombinant, non-recombinant, and unknown plasmid DNA respectively. This range is quite close compare to a typical concentration of plasmid DNA. For the 260/280 ratio, they are 1.93, 2.01, 1.92 from our group recombinant, non-recombinant, and unknown sample respectively. Also, the 260/280 ratio is 1.97, 1.78, 1.76 from our classmate recombinant, non-recombinant, and unknown sample respectively. In general, the ideally 260/280 ratio is from 1.8 to 2.0 range. Therefore, our group and our classmate group data are quite close compare to the ideally range

Key

Lane 1 DNA ladder
Lane 2 Double recombinant digest
Lane 3 Double non-recombinant digest
Lane 4 Double unknown digest
Lane 5 Single recombinant digest
Lane 6 Single non-recombinant digest
Lane 7 Single unknown digest
Lane 8 No recombinant digest
Lane 9 No non-recombinant digest
Lane 10 No unknown digest
Restriction Digest:

Figure 5: 0.9% agarose gel electrophoresis of restriction digests and undigested plasmid

Figure 5 shows the total of 9 digest reactions which consists Not I/ Nco I double digest, Nco I single digest and undigested for each of the three recombinants, non-recombinant and unknown plasmid DNA we isolated before. Unfortunately, in this restriction digest of plasmid DNA part of experiment, we used all recombinant, non-recombinant, and unknown plasmid DNA from our classmate group. No band was presented except DNA ladder in figure 5. According to 1.0kb DNA marker, 0.5kb band from DNA ladder which was loaded in our gel electrophoresis band should be the faintest band compare to other bands, but it looks like the brightest band. Also, 3.0kb band from DNA ladder should be the brightest band compare to others, but it looks quite very faint. Moreover, the visualization of the wells on the top is unproportioned. Some of those are very faint and smear, it looks overflow since we loaded too much sample into the well.

Key

Lane 1 10ul DNA ladder
Lane 2 Negative control
Lane 3 Positive control
Lane 4 Recombinant (1st group)
Lane 5 Non-recombinant (1st group)
Lane 6 Unknown (1st group)
Lane 7 Recombinant (2nd group)
Lane 8 Non-recombinant (2nd group)
Lane 9 Unknown (2nd group)
Gel Electrophoresis of PCR:

Figure 6: 0.9% agarose gel electrophoresis of PCR product

According to figure 6, lane 1 contains the DNA ladder which contains a standard length of DNA fragments. Lane 2 contains negative control which has one band at below 0.5kb. Lane 3 contains positive control which has two bands at about 1.1kb and below 0.5kb. Lane 4 and 7 contain recombinant plasmid. They both show same band at 1.1kb and below 0.5kb. However, a top band at 1.1kb in lane 4 looks brighter than lane 7. Lane 5 and 8 contain non-recombinant plasmid. Lane 5 shows only one band at below 0.5kb while lane 8 shows two bands at 1.1kb and below 0.5kb. Lane 6 and 9 contain unknown plasmid. They both show two band at 1.1kb and below 0.5kb. Moreover, we observe all the bottom band at below 0.5kb are faint and smear.

Virtual Cloning:

By using online tools, the PCR product size and the location of PCR primer determined. We found the PCR product which performed in gel electrophoresis of PCR experiment has 1183bp. Moreover, NcoI and NotI cut sites were determined to be 1401bp and 677bp respectively. The pET-41a(+) vector was also determined to be 5860bp while the the recombinant pET-41a(+)/EGFP insert had 6584bp.

Discussion:

Ligation:

The goal of this ligation part of the experiment is successful. In theoretical, pET41a(+) vector has 5860bp, and egfp insert has 724bp, so the circular recombinant pET-41a(+)/egfp plasmid DNA has 6584bp. Lane 2 and lane 3 showed a total of two bands. The top band at 6kb and smear above might be the open circular form of plasmid DNA. The bottom band at about 0.74kb presents the supercoiled DNA. Also, lane 3 contains more concentration egfp insert than lane 2, so the band at about 0.75kb in lane 3 looks brighter and bolder than lane 2. Lane 4 should have only one band at 6.584kb because it contains the circular recombinant pET-41a(+)/egfp plasmid DNA without DNA ligase. However, it shows a total of two bands at 10kb and 4kb in lane 4. The ligation #3 sample might be poor quality, it is contaminated cause smeared results in lane 4. Moreover, lane 5 contains sterile water only cause the result in the absence of bands. Lane 6 does not contain DNA ligase control, so the sticky end of egfp insert cannot seal and join pET41a(+) together. That is the reason why two bands are presented in lane 6. The top band is at 6kb should be represented as pET41a(+), and the bottom band is at 0.75kb should be represented as egfp insert.

Transformation:

The transformation recombinant plasmid into E. coli was successfully. According to figure 2, the presence of green and white colony is observed in plate 1 and 2. This is because they contain the recombinant pET-41a(+)/egfp plasmid on LB/Kan/IPTG medium. However, because of higher molar ratio of egfp insert in plate 2, there are more colonies in plate 2 compared to plate 1. Only 11 fluoresces green colonies under UV light are appeared in plate 3 because this plate contains circular recombinant pET41a(+) plasmid. White colony is not expected in this recombinant plasmid sample. Also, there is no colony in plate 4 because it contains only sterile water on LB/Kan/IPTG, so no bacteria or competent cells are growth in this plate depite of it is placed on LB/Kan/IPTG medium. There are only one white colony presents in plate 5 because this plate is missing IPTG, so Lac I repressor protein binds Lac operator and prevents transcription. Moreover, plate 6 observers a lawn of bacteria because of the absence of Kanamycin and IPTG in this plate.

For the transformation efficiency data, we can observe the TE data in plate 2 is higher than plate 1. This is also because of the higher molar ratio of egfp insert in plate 2 compare to plate 1. Miniprep:

Non-recombinant of our group data is lower than expected in figure 3. Buffer EB was added double in non-recombinant tube, this causes non-recombinant had double dilution lead to concentration of non-recombinant is reduce half as what expected. Moreover, a typical concentration of plasmid DNA yield from 50-100 ng/ul. Our group data is quite lower compare to a typical concentration. All three plasmid DNA samples might be more dilute during experiment because of uncertainty measurement volume through adding buffer step. Moreover, we also observe the absorbance of DNA at 260nm to the absorbance at 280nm. This indicates how purify of DNA in our sample of this miniprep of plasmid DNA experiment. The 260/280 ratio in our group non-recombinant sample is litter over compare to the ideally range, this is also because non-recombinant plasmid DNA is more diluted while buffer EB was added double in accidently.

Restriction Digest:

In theoretically, one band is shown in a single recombination digest plasmid at 6.584kb because the circular recombinant pET-41a(+)/egfp plasmid DNA has 6584bp. In double recombinant digest plasmid, two bands were expected to observe at 0.724kb and 5.86kb because the egfp insert has 724bp while pET-41a(+) vector has 5680bp. Also, in single non-recombinant digest plasmid should have one band at 5.933kb while a total of two bands should observe in double non-recombinant plasmid at 5.86kb and 0.073kb. Moreover, no band is expected to observe in both recombinant and non-recombinant undigested plasmid. However, the results in figure 3 wasn’t same as with what we expected. The uncertainty measurement amount of agarose leads the fragment ran too slow or quickly could be one of source of errors in this part of experiment. Also, in previous Mini prep part of experiment, all three non-recombinants, recombinant and unknown could be more dilute and contaminated are also the part of source of errors in this part of experiment.

Gel Electrophoresis of PCR:

We ran all PCR sample longer than expected, this leads to all the bottom band appear in all lane in this gel electrophoresis. One band was expected to observe in positive control and recombinant PCR sample. Also, no band was expected to observe in non-recombinant and unknown PCR sample. However, non-recombinant PCR sample in the second group showed one band at 1.1kb as same as recombinant PCR sample from two groups data, this result might be error because they might load recombinant PCR sample in the non-recombinant PCR well position, leads to get wrong result. Moreover, we started to run the gel electrophoresis at immediately 110V, this high voltage should lead bands in in figure 6 smeared. In the other hand, PCR sample might be contaminated. The poor-quality PCR samples were, the poor visualization showed in band.

References

“GNN – Genetics and Genomics Timeline.” GNN – Genome News Network, www.genomenewsnetwork.org/resources/timeline/1973_Boyer.php.
“Herbert W. Boyer and Stanley N. Cohen.” Chemical Heritage Foundation. N.p., 02 Sept. 2016.
Web. 13 Feb. 2019.

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