Jesse Ruben
Partner Roman Verner
BMB 442
Recombinants and Transformation
Introduction
The goal of this experiment was to take two antibiotic resistance genes for ampicillin and
kanamycin from plasmids pAMP and pKan and recombining them together. Then by
transforming this new plasmid into E. coli, the E. coli will then become resistant to these two
antibiotics. We then can calculate the efficiency of this experiment and percentage of
successful pKan plasmids made in molecules. Restriction Enzymes are used in order to break
the phospodiester bonds around the different antibiotic resistant gene on the two original
plasmids and a gel electrophorisis was made to make sure the DNA was broken. The broken
DNA has a stick end that is recombined at compatible ends of other broken fragments by a DNA
ligase and another gel was made to check the success of this process. This new multi-resistant
plasmid is transformed, pulled into the cell that is known not to have either gene and uses the
foreign plasmid, which now can express the resistance from transformed plasmid. Cells that can
transform a plasmid are normally are called competent, the ability to absorb outside plasmids,
but not all cells can do this. E.coli does not have competence, therefore a procedure is done so
that the membrane of the E. coli will be artificially competent-like, and absorb this multiresistant plasmid.
By plating the E. coli on LB-plates that contain the two antibiotics, colonies which grew
shows how many cells transformed this plasmid. Due to the high unpredictable chances that
the breakage and recombination of the DNA wasn't 100% accurate and other plasmids were
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made, or remained as pAMP and pKan, plates were used that contained only one antibiotic and
the colonies were then counted on these plates as well. These plating proved that our
procedure rendered the E. coli to become competent and transform the different plasmids. The
counted colonies are then used to find the percentage of successful transformation.
The first part of the lab required us to use two 1.7 ml mircofuge tubes to set up the
digestion for plasmids, pAMP and pKan, which contain the two desired antibiotic-resistant
genes want for recombination. By the use of both BamHl and HindIII restriction enzymes, the
two plasmids are digested and broken into two parts where these enzymes cut the backbone of
specific DNA sequence on a double strain DNA. The restriction enzymes leave two sticky ends
which can only be "glued" back together with a compatible sticky ends by the DNA ligase, which
is used later. 6ul of Each plasmid was then put into their individual tube and 1 ul of each
restriction enzyme was distributed into both tubes. Then 2.5 ul of 10x restriction buffer was the
add, which provided the restriction enzymes with a proper pH and ionic conditions to maximize
their activity. 14.5 ul of dd H2O was used to make each tube volume contain 25 ul. This mixture
was then incubated at 37oC for 35 minutes to complete the digestion reaction. After the
digestion 12 ul of our each digestion were separated and given 3 ul of 5x Sample buffer(total 15
ul) and for a 1.0% gel electrophoresis. The 15ul of each reaction was load onto the gel along
with a 10 ul of a DNA ladder for each partner. The last two wells were also loaded with 10ul of
the untreated pAMP and pKAN plasmid for comparison, to see if our digestions worked. The gel
was then photographed and the leftover of each digestion was then incubated to inactivate the
restriction enzymes, so the ligation process would work without being cut again.
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The ligation started by taking 7.5 ul of each digested pAmp and pKan into the one ligation
tube, along with 1 ul of DNA ligase and 3 ul of 10x ligase buffer, this provides proper conditions
to activate the ligase enzymes (total 19 ul). The mixture is then levels to 30 ul with 11ul of dd
H2O. This ligation mixture was then left in an ice bucket, which then is used for the
transformation and competence manipulation.
The strain HB101 E. coli is used because it's genome doesn't encode for Amp or Kan
resistance. Our goal was to transform the ligated plasmid into the cell by manipulating the
environment to induce E. coli to become competent, since this doesn't happen naturally for E
coli, we had to created the right conditions.
A prepared incubation of HB101 was given in a centrifuge tube and places in an ice
bucket, by keeping the cells cold their membranes motility slows down and allows for us
manipulate it. Then by centrifuging at 5,000 rpm for 5 minutes in a cold centrifuge, allowed us
to remove the supernatant and take the pellet of cells and resuspend them in 10 ml of ice-cold
CAST solution and placed on ice again. Due to the membranes naturally negative charge, the
plasmid that we are trying to transform will only be repelled, since DNA has a negative charge
as well. To fix this problem the CAST solution contains Calcium which is positively charged, this
can interact with the membrane allowing the DNA to pass by. Normally the membrane is so
motile that the Ca2+ has a hard time interacting with it, but since we keep the cells cold the
calcium was then able to interact with the membrane.
The mixture is then centrifuges again at 4000 rpm for 5 minutes and the supernatant is
completely removed again. We then resuspend the pellet with 1.2 ml of ice-cold CAST and
separate 0.2 ml of this mixture into four 12ml culture tubes labeled, one for each partner( 4ul
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of ligation DNA), one negative control (NO DNA added) and one positive control ( 2ul untreated
pKan DNA). The four tubes were then placed on ice for 20 minutes, which keeps the
membranes matrix slow. We then heat shocked the cells for 1.5 minutes at 42oC, at this point
the outside of the cell is warming up, but the inside of the cell is still cold. With the thermal
transfer opening up the membrane pores, the DNA plasmid is that was used in each tube was
(my ligation, partners ligation, pKan plasmid, No DNA) was also taken in. The cells are
immediately cooled, trapping the DNA inside of it, thus manipulating the competence of the
HB101 cell. The cells were then transferred into a culture tube of 0.8 ml of LB and incubated at
37oC for 45 minutes, this allowed the cells to recover by diluting the Ca2 and allowed them to
express the new plasmid they just absorbed.
By plating each reaction on a desired antibiotic plates, we can the count the number of
colonies grown and judge the complete transformation of our ligation and which resistance
was expressed. Each partner took 250ul of the transformed cells and placed them on the plate
with the Amp antibiotic, another with Kan antibiotic and one with both antibiotics to see if the
plasmid that was absorbed had one or both genes. 250 ul of the negative control with no DNA
was placed on the LB plate with AMP and another with KAN antibiotic to test if the antibiotics
even worked properly. And 100 ul of the positive control, which contained transformed pKan
DNA only, was placed on the pKan LB plate and LB plate with no antibiotics to count how many
cells were being plated, but first a serial dilution was made starting with 100 ul. After the final
dilution, 50ul of the 106 tube was plated on the normal LB plate, which was used to count the
number cells being plated and gives us the percentage of transformed efficiency.
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Another gel is then made, but at 0.8%, which is used to check our ligation product and
success, this gel was shared amongst 7 people and the ligation did work, which is mentioned in
the results. This means that some of the cells were being transformed with the correct plasmid
and would grow on the agarose plates with antibiotics.
Results
Gel Figure 1: After digesting the two plasmids we then did a ligation of the restriction
fragments. But before assuming that the DNA had been properly digested, we checked the
product with a gel Electrophoresis. Each Student placed 10 ul of a molecular weight DNA ladder
their before digestion samples. Then 15 ul of each digested reaction in the next wells, my
partner had placed his reactions in the 2nd and 3rd well and placed mine in the 5th and 6th well.
The last two wells contained 10 ul of the uncut plasmid pAmp and pKan, 7th and 8th well
respectively, which were
placed in the gel for
comparison for the reactions
to check if a proper digestion
took place. Table 1 (row A and
B) are rough size estimates of
the fragments were then
recorded using the DNA ladder
as a reference and Table 1 (row C) shows the actual base pair sizes in each of band of the well.
*There were different amount of bands that appeared in the uncut lanes of pKan and pAmp,
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which represented different topiosomers. The uncut pKan plasmids had two clear bands,
neither of them were the linearized topiosomer since the plasmid was uncut (this would have
appear as a third line between these two bands). The two bands that appeared were the
supercoiled plasmid, which traveled further due to its tightly packed matrix, and the nicked
topiosomer, which traveled slower due to its circular structure. The pAmp on the other hand
had a different result that still contained the supercoiled and the nicked topiosomer, but also
other topiosomers appeared above them. These other bands are due to plasmid multimers,
which are two or more plasmids that are linked together or chained around each other, this
results in a much slower distance traveled through the gel since more base pairs are attached.
* The BP size of the extra bands in lane 7 are multimer plasmids and can't be estimated
due to the type of gel used, which is meant for estimating lower base pair fragments
Table 1
Lanes
Estimated Band 1 Estimated Band 2 Actual/Expected sizes
A
B
C
Lane 1
DNA ladder
3755 bp
Lane 2
Romans pAmp
2333 bp
Lane 3
Romans pKan
4539 bp
Lane 4
DNA ladder
1,2,3,4…., 10 Kb
4194 bp
Lane 5 My pAmp Fragments
3800 bp
800 bp
3755 bp
784 bp
Lane 6 My pKan Fragments
2500 bp
1700bp
2333 bp
1861 bp
Lane 7
Uncut pAmp
3600 bp
10000 bp
4539 bp
///////
Lane 8
Uncut pKan
3100 bp
9500 bp
4194 bp
/////
The next step to analyzing the gel was to determine if both reactions were successfully
broken into two fragments, since each restriction enzyme breaks one specific site then two
fragments will be made. The pKan digestion was clearly broken and formed two bands different
from the plasmid band, but the pAmp digestion encountered a problem in determining if the
digestion was complete or not. One of the sizes of the fragment’s were 3755 base pair long and
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had traveled close to the same distance of the supercoiled plasmid of pAmp, around 3800 base
pair. The solution can be deduced first by determining if the plasmid was only partial digested
by only one restriction enzyme then the plasmid would be linearized and travel slower than the
supercoiled plasmid which would clearly produce a distinguishable band, in this experiment we
did not have a partially digested plasmid. Now we must determine if neither enzyme worked
and if it left a supercoil band, which produces the problem, since the fragment of pAmp
traveled the same distance as the supercoil plasmid. Visually this can’t be distinguished, but by
deducing the fact that there were no linearized plasmid would indicates that either both
restriction enzymes work or that neither of the enzymes worked. Clearly we had the second
band from the digestion, at 784bp, which means most likely the digestion did work and all of
the enzymes did do their job correctly. So all of the fragments agreed with the predicted
positions made from the actual size of each fragment and plasmid.
After completing the first gel to make sure the digestion took place we then preformed a
ligation of these our two digestions by mixing them together and using the protocol mentioned
in the introduction. Before proceeding with the transformation process, my partner and I first
preformed the second gel, along with partners of other groups, to check if the ligation was
successful. The gel showed new large DNA plasmids, but this doesn’t tell us if we produced the
desired plasmid with both genes. Gel figure 2:
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lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
DNA ladder
My Ligation
Romans Ligation
Dannys Ligation
Jennys Ligation
X ligations
Y ligation
Z ligation
The plasmids traveled all along the gel and created multiple bands, which meant that
the ligation had happened and different fragments connected together creating a diverse
number of plasmids. The diversity was based on the number of fragments that have been glued
together creating a giant circle, and/or plasmids that were looped together forming a chain.
This diversity was a clear indicator that the ligation had worked, but there were some
fragments that had missed the ligation, which are indicated by having less base pairs and still
traveled further down the gel. As expected ligation wasn’t 100% successful in producing the
desired multi-resistant plasmid we were trying to create, so we must take the ligation reaction
and transform them into a cell to find out how many desired plasmids were made. If the
ligation didn’t work we would expect to see the still digested fragments in gel figure 1, but in
one well.
After checking the success of the ligation we then manipulated the E. coli HB101 into
competent state, which would absorb the ligated plasmids and express the proteins for the
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resistance to either or both antibiotics. The cells grew on LB plates with an antibiotic,
depending on the uptake transformed of plasmid; the cell would grow or die on the plate with
one or both antibiotics. The procedure is mentioned in the introduction and Table 2 indicates
the recorded number of
Type of plate
KAN
AMP
Amp/Kan
LB
21
15
10
----
The negative control was made
Jesse's
Ligation
to eliminate any possibility of
Romans's
Ligation
30
12
8
----
No DNA
(Negative Control)
0
0
----
----
pKan
(Positive Control)
261
----
----
57
colonies grown on each plate.
corruption in the antibiotics, so
E. coli HB101 with neither
resistant gene didn't grow on
any antibiotic plate, which indicates that both antibiotics worked and we didn’t count any E.
coli without the proper genes on a specific plate. The positive control was made so that we can
calculate the efficiency of transformation and the percentage of successful plasmids made. The
efficiency was calculated by the number of transformed bacteria, positive control KAN plate
which was serial diluted to 106, divided on the by the total number of bacteria plated, positive
control LB plate: 261 colonies of pKan E. coli were counted on the Kan-LB plate of 250ul and 57
colonies of pKan E .coli were counted on the 106 serial dilution LB plate of 100 ul.
Transformation effiency =
e=
=
.000018315
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.0018%
Another way to represent the efficiency of the plasmid is to calculate the
percentage of successful plasmids made. By converting the base pair of the pKan plasmid,
4194 bp, into gram per moles, 1 bp =
then by multiplying the amount of grams
put into the reaction, 2ul of 5ng/ul, we can then finding out how mole of pKan were put
into the transformation reaction and convert that into molecules.
4194 bp x
10ng x
=
4.335 x 10-15 moles
4.335 x10-15 moles x 6.022 x1023
=
2,610,537,000 molecules
Then by multiplying the percentage plated to the number of total molecules added
from the start, we then can calculated the total number of pKan molecules in the plated
transformation.
2,610,537,000 mol. x
=
= 815,792,812.5
Total pKan mol. plated
Now by dividing the number of successful plasmids (counted on pkan plate) by the
total pKan molecules plated and multiply by 100% we can then calculate the goal of this
experiment, which is instead of just calculating the percentage of cell that transformed
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and grew, we also calculated percentage of molecules that were transformed into the
cells as well.
x 100% =
3.199 x 10-5 % of successful plasmids
Discussion
The counted cells on each antibiotic plate had very close numbers of cells that
absorbed a plasmid with one or both of the resistance genes. The known hypothesis of
Recombination and Transformation is that the origin of replication (ORC) gene sequence
allows the plasmid to be functional and expressed in the cell. The fragment that contains
pKan doesn't have the ORC gene, but the pAmp fragment does have the ORC gene.
Therefore whichever plasmid pAmp is ligated to will also have function, unlike pKan
which needs to ligate to a fragment that contains ORC, in order for KAN resistance to be
expressed. According to this understanding pAMP will be expressed more often than
pKan and the likelihood that pAmp and pKan fragments ligated together expressing both
is a 4:2:1 ratio. In this experiment the pAmp resistance did appear more frequently than
pKan, and the plasmid with both resistances happened less often as well, the frequency
was a 21:15:10
4:3:2, which are very close to hypothesized frequency.
If we wanted to get a higher chance of obtaining cells with only desired plasmid, a
technique of gel purification could had been done. By slicing out the desired digested
fragments in the gel and melting the gel. We then could purify the fragments out of the
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gel and perform a ligation only between the fragments with pKan and pAmp. The ligation
reaction would have been in favor of creating only our multi-resistant plasmid and we
then could transform them into the HB101 cells.
The numbers calculated to get the transformation efficiency and percentage of
successful plasmids tells us that the transformation of the plasmid is a very hard process
to overcome on a cell that needs to be manipulated for competence. If the conditions
stayed the same and more DNA was used then more cells would have transformed and
the transformation efficiency and percentage of successful plasmids would
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