Biology

The effect of UV light on the CaCl2/PEG 3350/DMSO transformation buffer for CRISPR Cas9-edited E. coli

Abstract

Bacterial transformation is a method in which the composition of a bacterial genome is altered via uptake of DNA from its external environment. This experiment aims to understand the role of the transformation buffer in the CRISPR-Cas9 system, with specific focus on the Polyethylene Glycol (PEG) component. A DIY CRISPR kit containing PEG in the transformation buffer modifies the DNA of E. coli bacteria. PEG is an essential part of the buffer, although its actual function in the process of preparing bacterial DNA has not yet been elucidated. For example, it is suspected that PEG plays a role in allowing the DNA to move past the bacterial cell wall, given that both are negatively charged and so will repel. PEG, along with calcium chloride, may be used to neutralise charges. It also may have functions in facilitating DNA transport into the cell and making the cell membrane more porous. To determine the specific role of PEG, the protocol aims to degrade the compound, thus removing it from the buffer itself. After this, the final effects on the transformation result will be observed in order to identify any changes caused. This will be done via treatment with heat and UV light, which only affects the PEG component. Therefore, it is assumed that changes in the final CRISPR DNA transformation are due to the absence of PEG. Previous literature shows that degradation by both heat and UV alters the structure of PEG¹,², and that PEG 3350 and PEG 8000 are most suited to facilitate DNA entry into the cell. Therefore, it can be assumed that the gradual degradation of PEG would eventually inhibit the whole CRISPR-editing process. Results showed that the degradation of PEG slowed down the process considerably, reaffirming the conclusions found in the papers cited.

Introduction

In recent years, many articles on CRISPR-mediated gene editing have been published, with particular focus being in the application of this gene-editing technology in cancer research. However, recent reports indicate that CRISPR-edited cells may trigger a DNA damage response dependent on the cell cycle protein p53, stopping cell growth. Overriding this response would make CRISPR more efficient; however, the p53 protein is also involved in the prevention of cancer. The data suggests that the increased cancer risk stems from the transplantation of CRISPR-edited cells into patients with inherited diseases3. The aim of this investigation was to develop understanding of the underlying CRISPR mechanism via a do-it-yourself CRISPR Cas9 kit purchased from The Odin Company (2018).

What is CRISPR ?

CRISPR is an acronym standing for “Clustered Regularly-Interspaced Short Palindromic Repeats”, and encompasses a family of bacterial DNA sequences that repeat at regular intervals4. CRISPR technology is based on the natural defense mechanisms of these microbes against bacteriophages and viruses. When foreign DNA from another organism that has not previously been encountered is injected into the cell, the CRISPR immune system thwarts the attack by destroying the genome of the invader5. This is done by cutting out the DNA section using what has been identified as CRISPR associated genes (or Cas genes), which code for Cas enzymatic proteins. In the CRISPR-Cas9 system, Cas9 is the enzyme that acts like a pair of molecular scissors and makes a precision cut on the foreign DNA based upon a pattern. If the cell encounters foreign DNA, a piece of that DNA is saved in the segments between the aforementioned repeated DNA clusters. These segments are called spacer DNA. Much like a collection of keys on a keychain, the next time a bacteriophage attacks, the bacteria checks the saved “key” for a match against the phage DNA. If there is a match, the bacteria can then transcribe a CRISPR RNA (crRNA) sequence to target that specific pattern and “cut” it out immediately using the Cas enzyme, thus stopping the spread of the virus. If the foreign DNA has not encountered before, a new spacer is generated, adding a new key to the keychain to be used in secondary infection responses6. Figure 1 illustrates the process. Thus, this spacer chain becomes a history of the various attacks on the cell, since the new spacers are added sequentially with the most recent DNA nearest to the leader sequence.

Figure 1- A diagram depicting the CRISPR system in bacteria defending against future infection. (Chu, K. 2019)

In 2012, two important research papers7,8 concluded that Cas9 could be used to cut any region of DNA using “guide RNA” (gRNA), transforming the CRISPR-Cas9 system into a simple, programmable genome editing tool. Once the DNA is cut, a DNA template can be used in conjunction with the cell’s natural repair mechanism to edit the genome. Since then, many articles and papers have come out demonstrating multiple uses for CRISPR-Cas9 gene editing technology in a variety of different industries.

Components of the CRISPR-Cas9 gene editing system:

  • Cas9 protein to cut the genome
  • gRNA to guide the Cas9 to the user defined site; it is the combination of two types of RNA: tracrRNA (trans-activating crRNA) and crRNA (CRISPR RNA). crRNA is shown in Figure 1.
  • Optional template DNA in case a specific gene sequence is to be programmed

In this experiment, the gRNA and template DNA induce a substitution mutation at the base position coding for the 43rd amino acid in the sequence, changing a lysine to a threonine residue. This change encodes for a gene conferring resistance to the antibiotic streptomycin, allowing E. coli to survive on Strep media instead of inhibiting its growth.

Experiment

Protocols for preparation of agar plates and Strep media, and preparation of bacterial cells for transformation are included in the Appendix. The experiment involves three separate trial9. The aims are as follows:

  • Trial 1: To confirm the contents of the kit and show that E. coli will grow on the agar plates but not on the LB Strep plates.
  • Trial 2: To confirm that the CRISPR-Cas9 system could edit the DNA of E. coli and to define variables related to the transformation buffer that may influence the growth rates of E. coli.
  • Trial 3: To perform a more systematic experiment by controlling the variables

based on the trial 2 results; if the results of trial 2 are inconclusive, the trial should be repeated more carefully or changed to expand the variable space.

Trial 1

This first trial confirms the contents of the CRISPR Cas9 system kit, and allows validation of the procedures and methods used. In order to create a successful experiment, the procedures must be systematic, precise and reproducible. Tests aim to verify that the E. coli bacteria samples grow, and that LB Strep media inhibits growth.

Procedure

Two LB Agar Media plates and two LB Strep/Kan/Arab Agar Media plates were prepared and refrigerated overnight. A sample of E. coli was inoculated onto all four media plates using a sterile loop and incubated for 24 hours.

Results

Table 1 displays the growth of unedited E. coli on LB Agar Media and LB Strep/Kan/Arab Agar Media plates over 24 hours. This was done as a control sample for further experiments.

Table 1- Qualitative description of growth of E. coli strains under fixed conditions on two agar media.

Time (hrs) LB Agar Media 1 LB Agar Media 2 LB Strep/Kan/Arab Agar Media 1 LB Strep/Kan/Arab Agar Media 2
0 hours No growth No growth No growth No growth
24 hours Lots of growth Lots of growth No growth No growth

Discussion

The unedited E. coli did not grow on the LB Strep/Kan/Arab Agar Media plates because this strain of E. coli is sensitive to streptomycin. The E. coli that grew on the LB Agar Media plates is used later in the experiment.

Trial 2

Review of previous papers provided better understanding of the role of PEG in the transformation buffer10,11. Higa (1970) demonstrated successful DNA transformation in E. coli by using a chemical method involving the bacteria and DNA in a suspension of cold CaCl2; the effectiveness of this technique has informed many of the protocols used in subsequent papers. Chung (1989) explored the effect of different chemical components of the transformation mix on the transformation efficiency. By varying the composition of the transformation mix (excluding some components and including others), the authors showed that a solution of LB Agar, DMSO(Dimethyl Sulfoxide), PEG 8000, and Mg2+ had the highest transformation efficiency out of the 8 different mixtures that they investigated. Chung highlights transformation efficiency as a precise metric that is used to measure the number of DNA molecules that have been changed. For the purposes of this experiment, growth rate of the edited E. coli with respect to time will be used as the metric.

The composition of the transformation buffer used is as follows: LB Agar, 25mM CaCl2, 10% PEG 3350 (Polyethylene glycol 3350) and 5% DMSO. The salt component (CaCl2 instead of Mg2+) in the buffer is different to that used in the Chung experiments, but other chemical components and concentrations are kept the same. Due to the ionic nature of either salt, it is highly probable that its primary function is to neutralize the negative charge either on the DNA12 or the bacterial cell walls to facilitate transport of DNA into the cell13.

One other dependency that Chung’s paper highlighted was a dependency of the transformation efficiency on the molecular weight of PEG. The highest transformation efficiency occurs when the molecular weight is between 3350 and 8000 and drops off very quickly outside that range particularly at low molecular weights. One of the suggested explanations of the role of PEG in the CRISPR kit “Since both DNA and cell walls are negatively charged, they reject each other. PEG 3350 is thought to function by shielding the charge of the DNA, thereby making it easier to permeate the cell wall. PEG 3350 is also thought to help transport the DNA into the cell, as well as make the cell membrane itself more porous.” 11

Procedure

The bacteria transformation buffer was altered by exposure to high temperatures and UV light. Experimentally, more accurate data could be achieved if necessary equipment for the separation and purification of PEG 3350 was available. The assumption made during this protocol was that the CaCl2 and DMSO components are less affected by temperature and UV than PEG, as CaCl2 is a simple salt compound. Further experiments would be required to confirm that the DMSO is also not substantially affected by the exposure. Methods of UV and heat degradation were chosen based on previous work demonstrating fundamental changes to PEG structure after exposure to increased temperatures2 and UV irradiation1. By examining the growth rates of edited E. coli on the streptomycin growth plates, changes in the effectiveness of PEG due to temperature and UV exposure can be determined. It may be possible to isolate the specific property of PEG responsible for enhancing the transformation efficiency by comparing the results. In order to ensure safe interaction of UV light with the buffer without heating the sample itself, a PVC pipe housing was constructed.

The aim of trial 2 was to determine whether the application of UV light, heat, or both, would impact the growth rate of edited E. coli on LB Strep/Kan/Arab Agar. See Table 2.

Table 2- Trial 2 Simple Split Table

Split 0 1 2 3 4 5
Media LB Agar LB/Strep Agar
Heat no no no yes yes no
UV no no yes yes no no

In order to expose samples of PEG 3350 (via the transformation buffer), a safe and reliable UV apparatus was created.

UV Apparatus

  • 40 Watt UV lamp (wavelength 245 nm) with power supply (used for ponds)
  • 1 piece of 2” diameter PVC pipe
  • Clamps
  • Saw
  • Drill with different size bits
  • Wire
  • Fan
  • Thermometer
  • Object with surface at least 2” by 2”
  • Timer/stopwatch
  1. Clamp the PVC pipe. Saw off part of the PVC pipe so that it is roughly 2 feet longer than the UV lamp(See Image 1 for details).
  2. Drill two small holes near the ends of where the lamp would be once it is placed inside the PVC pipe (See Image 2).

Image 1- PVC preparation for UV apparatus – Cut to length

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Image 2- PVC preparation for UV apparatus – Drill holes for wires

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  1. Drill one large hole at one end of the pipe. Then, using the saw, cut out a slit. This will be where the UV lamp cord goes. See Image 3.
  2. Place the UV lamp into the PVC pipe. Use the wires to hold it in place. The lamp should be suspended, not resting at the bottom of the pipe. See Image 4.
  3. Place a fan at one end of the pipe to complete the setup. The need for the fan will be explained later. See Image 5.

Image 3- Drill hole and cut slot for power cord

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Image 4 – UV Lamp placement in PVC tube

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Image 5 – Completed UV apparatus setup

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The sample holder was made from a contact lens case with the covers removed, which was then attached to a flat, narrow section of wood. This would facilitate sliding the sample in and out of the apparatus and expose the transformation mix to UV light.

The duration and intensity of both parameters (that is, the length of UV exposure and the temperature of applied heat) had to be considered, given that the criteria for resulting perceptible changes in growth rate had not been defined yet. The scope of the experiment would have to be widened to accommodate for this discrepancy, but if the conditions were too harsh, secondary effects or confounding variables could affect the accuracy of the data.

As seen in Chung’s paper, the valid temperature range used for heat-shocking the bacteria in preparation for transformation of genetic material was from 42 – 75°C; thus, 75°C was used as the maximum limit in this experiment. The heat shock component is typically applied after the transformation buffer is added to the Cas9 mixture, to ensure maximum uptake of plasmid DNA. However, for the purposes of this experiment, the temperature is applied to the transformation buffer only.

For the UV exposure, it was difficult to determine a starting point for the time. Previous literature used times ranging from 1 hour to 7 hours, with a 150W UV source and 100ml samples1. The sample size in this experiment was 1000 times smaller in volume, and the UV source is roughly 1/4 less power. Thus, it was assumed that 6 minutes of UV exposure would be sufficient for a significant change in PEG content.

Results

Table 3 – Trial 2 Results

Split Condition 0 1 2 3 4 5
Media LB Agar LB/Strep Agar
Heat (@71°C) Duration No heat No heat No heat 1 minute 1 minute No heat
UV (@40W) Duration No UV No UV 6 minutes 6 minutes No UV No UV
Cas9 Yes Yes Yes Yes Yes No
Time Elapsed (hours) Split Condition
0 1 2 3 4 5
0 No growth No growth No growth No growth No growth No growth
7 Completely full of growth 25 specks of white growth (E. coli) 4 specks of white growth (E. coli) No growth No growth No growth
12 Still completely full of growth (more than before) 27 slightly larger specks of white growth – includes one large speck (E. coli) 4 slightly larger specks of white growth (E. coli) No growth No growth No growth
19 Still completely full of growth (more than before) 27 larger specks of white growth – large speck also getting larger (E. coli) 5 slightly larger specks of growth (E. coli) No growth No growth No growth
24 Still completely full of growth (more than before) 27 larger specks of white growth – large speck also getting larger (E. coli) 5 slightly larger specks of growth (E. coli) No growth No growth Other white fuzzy growth (definitely not E. coli)
30 Still completely full of growth (more than before) 27 larger specks of white growth – large speck becoming whiter and larger (E. coli) 5 specks of growth that are roughly the same size as the previous time (E. coli) Other white fuzzy growth (definitely not E. coli) Other white fuzzy growth (definitely not E. coli) Other white fuzzy growth (definitely not E. coli)

Figure 2 – Growth Sites versus Time (Trial 2)

Note that there is complete overlap of some curves on the graph and thus not all data are visible. Data shown in Figure 2 is altered to show highest relevance.

Discussion

Evaluation of procedure

Initial tests utilised 2” PVC end caps to seal the tube so that the UV light was contained within the apparatus. However, the 40W UV lamp gave off sufficient heat to degrade the rubber bands used to suspend the UV lamp. Therefore, the rubber bands were replaced with more durable wire, and a thermometer was used to measure the temperature both outside and inside the PVC pipe when the UV lamp was turned on. In order to minimize the unintentional heating of samples during UV exposure, a fan was placed at one end of the tube to create airflow for a cooling effect through the tube. The opposite opening was also blocked to avoid exposure to UV light for safety precautions, while still allowing the air to flow through the tube and maintain constant internal and external temperatures.

Analysis of results

The results seen in split 0 are from split 1’s leftover CRISPR-edited bacteria grown on a LB Agar media plate, rather than a LB Agar Strep/Kan/Arab Media plate. This split was ran in order to confirm that the edited E. coli that was used for the rest of the splits were viable. If the edited E. coli did not grow on the LB agar plate, it would be an indication of errors in carrying out the protocol to prepare the transformation buffer. However, this plate produced the most growth, which was a good indicator that there was not a significant error in the sample preparation. The rest of the data are the results from growing the edited E. coli on LB Agar Strep/Kan/Arab media plates. Splits 1 and 5 were controls, so their transformation mixes were not treated by UV or heat. The transformation mix in split 2 was exposed to UV light (40W for 6 minutes), while split 3 was exposed to both UV light (40W for 6 minutes) and heat (71C for 1 minute). Split 4 was exposed only to heat (71C for 1 minute). Split 1 showed far less growth than that of the control (Split 0), although split 1 still had the second highest number of growth sites visible. Split 2 had the third most growth. Splits 3, 4, and 5 all had no E. coli growth whatsoever.

During the sample preparation, there was not enough Cas9 and gRNA for both splits 4 and 5; thus, the results in split 5 are an underestimation of the true value. Split 5 was essentially the same as 1 except that it received far less gRNA and no Cas9. If it had received enough gRNA and Cas9, in theory, it is very likely that there would have been growth on that plate. It is uncertain, however, whether receiving less Cas9 affected split 4.

Nonetheless, there are several conclusions from the data we can draw.

The main conclusion from trial 2 is that exposing the transformation buffer to UV light does impact the growth rate of edited E. coli, since the number of E. coli growth sites for split 2 (UV only) and split 3 (UV and heat) is much lower than that of the control group (see Figure 1). This is significant because the ability for UV light to change may be one of the elements necessary to make competent bacteria, that is, bacteria than can take up DNA from the external environment.

Another conclusion from splits 1 to 5 is that the CRISPR Cas9 procedure for the DNA editing of E. coli for this experiment was successful; the edited E. coli were able to grow on the LB/Strep media, while in Trial 1, unedited E. coli were not. Also, a comparison between split 1 (control) and split 5 (control but without Cas9) shows a significant difference in growth sites. Although it is expected that without the Cas9, split 5 should show similar results for the unedited E. coli on LB/Strep media in trial 1, these results validate the components of the CRISPR kit and confirm accurate implementation of the protocols.

Lastly, the results for heating the transformation matrix were inconclusive, due to a shortage of materials available to generate sufficiently significant data for split 5. Split 3 (heat and UV) and split 4 (heat only) showed no E. coli growth after 24 hours, an indication of the negative effect of heat on the transformation matrix with respect to transformation efficiency. However, the decreased volume of Cas9 solution used may have been another confounding variable. Split 3 may have showed no growth due to some variation in UV exposure if 6 minutes is very close to the transition point; further investigation would be needed to confirm this, as was done in Trial 3 with the expansion of the parameter range.

Trial 3

The aim of this trial was to perform a more systematic experiment by controlling and changing the variables based on the results of trial 2. Since trial 2 did show that irradiating the transformation buffer with UV light does change its effectiveness on the edited E. coli growth rate on LB/Strep media, Trial 3 involved running several more splits on UV exposure to narrow down the range of duration times at which growth rate initially changes. From this, the cause of the change in E. coli growth rate may be determined depending on changes in the transformation buffer, and in particular, PEG 3350 under specific conditions. Since the application of heat showed inconclusive results, the splits were repeated and an extra split was added to expand the range.

Procedure

Trial 3, as seen in Table 2 and graphically in Figure 1, demonstrated that 6 minutes of UV exposure was a sufficient length of time to change the properties of the transformation matrix enough to impact the growth rate of edited E. coli on LB Agar Strep/Kan/Arab media. In order to better profile the transition time needed, splits were run at intervals of time instead of for a full 6 minutes of UV exposure (0, 3, 6, 9, and 12 minutes). For the heat application, the previous protocol in Trial 2 was repeated, with an additional split at 80°C for 1 minute. See Table 4.

Table 4- Trial 3 Split Table

Split 1 2 3 4 5 6 7 8
Minutes
Control 0 X X
UV (40W) 3 X
4 X
9 X
12 X
Heat (70˚C) 1 X
Heat (80˚C) 1 X

There was enough UV/heat treated transformation buffer to complete a second run and determine whether the results were repeatable, as well as if additional time was necessary to recover and replicate the DNA for the CRISPR engineering process. This step was highlighted in the CRISPR protocols as being very important.

Results

Table 5- Trial 3: Run 1

Time (hours) Plate Description
1 (control) 2 (3 min UV) 3 (6 min UV) 4 (9 min UV) 5 (12 min UV) 6 (70˚C) 7 (80˚C) 8 (control)
0 No growth No growth No growth No growth No growth No growth No growth No growth
10.5 Tiny white spots (E. coli) Tiny white spots (E. coli) Tiny white spots (E. coli) Bacteria streaks slightly paler Bacteria streaks slightly paler Bacteria streaks slightly paler White spots at end of streak (E. coli) Bacteria streaks slightly paler
18.5 Lots of growth (E. coli) Tiny white spots (E. coli) Lots of growth (E. coli) Tiny white spots (E. coli) No growth Tiny white spots (E. coli) Medium amount of growth (E. coli) Medium amount of growth (E. coli)
22.5 Lots of fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More tiny white spots (E. coli) Lots of fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More tiny white spots (E. coli) No growth More tiny white spots (E. coli) Lots of growth but not as much as on 1 & 3 (E. coli) Lots of growth but not as much as on 1 & 3 (E. coli)
25 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More larger white spots (E. coli) with some fuzz on edges (possibly E. coli) More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More larger white spots (E. coli) with some fuzz on edges (possibly E. coli) No growth More larger white spots (E. coli) Lots of growth – about as much as on 1 & 3 (E. coli) Lots of growth – about as much as on 1 & 3 (E. coli)
30 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More larger white spots (E. coli) with more fuzz on edges (possibly E. coli) More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More larger white spots (E. coli) with more fuzz on edges (possibly E. coli) No growth More larger white spots (E. coli) Lots of growth – more than on 1 & 3 (E. coli) Lots of growth – more than on 1 & 3 (E. coli)
34.5 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More white growth (E. coli) with more fuzz on edges (possibly E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) More white growth (E. coli) with more fuzz on edges (possibly E. coli) Very sparse tiny white spots (E. coli) More white growth – more than on 2 & 4 (E. coli) Lots of growth – more than on 1 & 3, concentrated around edges of streak (E. coli) Lots of growth – more than on 1 & 3 (E. coli)
36.5 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More white growth (E. coli) with more fuzz on edges (possibly E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) More white growth (E. coli) with more fuzz on edges (possibly E. coli) Very sparse tiny white spots (E. coli) More white growth – more than on 2 & 4 but less concentrated than on 7 (E. coli) Lots of growth – more than on 1 & 3 but less than on 8, concentrated around edges of streak (E. coli) Lots of growth – very concentrated, more than on 1 & 3 (E. coli)
42.5 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More white growth (E. coli) with more fuzz on edges (possibly E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) More white growth (E. coli) with more fuzz on edges (possibly E. coli) Very sparse tiny white spots (E. coli) More white growth – more than on 2 & 4 but less concentrated than on 7 (E. coli) with some fuzz on edges (possibly E. coli) Lots of growth – more than on 1 & 3 but less than on 8, concentrated around edges of streak (E. coli) Lots of growth – very concentrated, more than on 1 & 3 (E. coli)
46 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) Less fuzzy growth than on 1 & 3 (possibly E. coli) but lots of white growth (E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) Less fuzzy growth than on 1 & 3 (possibly E. coli) but lots of white growth (E. coli) Very sparse white growth (E. coli) Less fuzzy growth than on 2 & 4 (possibly E. coli) but lots of white growth – more than on 2 & 4 (E. coli) Lots of growth – more than on 1 & 3 but less than on 8, concentrated around edges of streak (E. coli) Lots of growth – very concentrated, more than on 1 & 3 (E. coli)
48 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) Less fuzzy growth than on 1 & 3 (possibly E. coli) but lots of white growth (E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) Less fuzzy growth than on 1 & 3 (possibly E. coli) but lots of white growth (E. coli) Very sparse white growth (E. coli) Lots of growth – very concentrated, slightly less than on 8 (E. coli) Lots of growth – very concentrated, slightly less than on 8 (E. coli) Lots of growth – very concentrated, more than on 1 & 3 (E. coli)

Table 6 – Trial 3: Run 2

Time (hours) 1 (control) 2 (3 min UV) 3 (6 min UV) 4 (9 min UV) 5 (12 min UV) 6 (70˚C) 7 (80˚C) 8 (control)
0 No growth No growth No growth No growth No growth No growth No growth No growth
10.5 No growth No growth No growth No growth No growth No growth No growth No growth
18.5 Lots of tiny white spots (E. coli) Lots of tiny white spots – slightly smaller than on 1 & 3 (E. coli) Lots of tiny white spots (E. coli) White growth on edges of streak (E. coli) No growth Very few tiny white spots (E. coli) No growth Lots of tiny white spots – slightly smaller than on 1 & 3 (E. coli)
22.5 Lots of fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More tiny white spots – more than on 4 & 6 (E. coli) Lots of fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More tiny white spots (E. coli) No growth More tiny white spots (E. coli) Lots of growth but not as much as on 1 & 3 (E. coli) Lots of growth but not as much as on 1 & 3 (E. coli)
25 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More larger white spots – larger but more sparse (E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) More larger white spots – smaller but more concentrated than 6 (E. coli) No growth More larger white spots – smaller but more concentrated than 2 (E. coli) Lots of growth – about as much as on 1 & 3 (E. coli) Lots of growth – about as much as on 1 & 3 (E. coli)
30 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) Lots of growth – less than on 4, larger specks (E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) Lots of growth – less than on 6, more sparse (E. coli) No growth Lots of growth – less than on 7, more sparse (E. coli) Lots of growth – less than on 8 (E. coli) Lots of growth – more than on 1 & 3 (E. coli)
34.5 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) Lots of growth – less than on 4, larger specks (E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) Lots of growth – less than on 6, larger specks (E. coli) Very sparse tiny white spots (E. coli) More white growth – more condensed than on 2 & 4 (E. coli) Lots of growth – less than on 8 (E. coli) Lots of growth – more than on 1 & 3 (E. coli)
36.5 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More white growth – less than on 4 (E. coli) with more fuzz on edges (possibly E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) More white growth – less than on 6 (E. coli) with more fuzz on edges (possibly E. coli) Very sparse tiny white spots (E. coli) More white growth – more than on 2 & 4 but less concentrated than on 7 (E. coli) Lots of growth – more than on 1 & 3 but less than on 8 (E. coli) Lots of growth – very concentrated, more than on 1 & 3 (E. coli)
42.5 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) More white growth – less than on 4 (E. coli) with more fuzz on edges (possibly E. coli) More fuzzy growth on edges – a lot more than on 1 (possibly E. coli) & more white growth (E. coli) More white growth – less than on 6 (E. coli) with more fuzz on edges (possibly E. coli) Very sparse tiny white spots (E. coli) More white growth – more than on 2 & 4 but less concentrated than on 7 (E. coli) Lots of growth – more than on 1 & 3 but less than on 8 (E. coli) Lots of growth – very concentrated, more than on 1 & 3 (E. coli)
46 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) Less fuzzy growth than on 1 & 3 (possibly E. coli) but lots of white growth – less than on 4 (E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) Less fuzzy growth than on 1 & 3 (possibly E. coli) but lots of white growth – less than on 6 (E. coli) Very sparse white growth (E. coli) Less fuzzy growth than on 2 & 4 (possibly E. coli) but lots of white growth – more than on 2 & 4 (E. coli) Lots of growth – more than on 1 & 3 but less than on 8 (E. coli) Lots of growth – very concentrated, more than on 1 & 3 (E. coli)
48 More fuzzy growth on edges (possibly E. coli) & more white growth (E. coli) Less fuzzy growth than on 1 & 3 (possibly E. coli) but lots of white growth (E. coli) More fuzzy growth on edges – more than on 1 (possibly E. coli) & more white growth (E. coli) Less fuzzy growth than on 1 & 3 (possibly E. coli) but lots of white growth (E. coli) Very sparse white growth (E. coli) Lots of growth – very concentrated, slightly less than on 8 (E. coli) Lots of growth – very concentrated, slightly less than on 8 (E. coli) Lots of growth – very concentrated, more than on 1 & 3 (E. coli)

Figure 3- Examples of E. coli Growth

No growth Some growth Moderate growth

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Lots of growth Most growth

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Discussion

Trial 3 resulted in a much higher yield for the CRISPR-edited E. coli. In trial 2, the growth sites were easily countable, but in this trial, it was much more difficult as many of E. coli growth sites started to merge together. This is most likely due to two possible causes. The first is that the perishable components (non-pathogenic E. coli bacteria, Cas9 plasmid Kanr, gRNA plasmid Ampr, and template DNA) were all from a different batch since the components had to be replenished for the third trial, and it is possible that this batch was fresher. The second and more likely cause is that the second repeat of the protocol was more accurately followed and the techniques used were more precise. However, the layout of the data presentation could be improved if there was a more quantitative way to measure the transformation efficiency as shown by Chung (1989) to create plots of growth sites versus time11.

From both runs in Trial 3, 12 minutes of UV exposure for the transformation buffer is almost enough to suppress the transformation of E. coli completely. Lower exposure times result in more E. coli growth sites shown.

Heat treatment of the transformation buffer was also inconclusive. While the second run seems to indicate a slower growth rate for 70°C and an even slower growth rate for 80°C when compared to no heat treatment, there are no consistent results from the first run. Therefore, it is possible that the duration of heat treatment needs to be longer; the time specified in the protocol may have been too short so that the temperature of the mixture did not reach a sufficient threshold inside the plastic microcentrifuge tube.

Conclusion

This experiment demonstrated that 12 minutes of UV exposure for the transformation buffer (10% PEG 3350, 5% DMSO, 25mM CaCl2) is almost enough to completely suppress the transformation of E. coli. While the effect of heat treatment 70°C (1 minute) and 80°C (1 minute) on the transformation buffer was essentially inconclusive, there did appear to be some effect. The initial aim was to vary these two parameters and analyze which properties of PEG are significant to the transformation efficiency, since UV and heat affect PEG differently. However, since only the UV light exposure seems to have yielded consistent results, one can only speculate that molecular weight plays a role, since UV irradiation has been shown to degrade PEG and decrease the molecular weight1. Perhaps these long chains of PEG somehow shield the negative charge of the DNA, thereby minimizing the repulsive force of the negatively charged cell wall. When the long chains are shortened after UV exposure, it is possible that it becomes less effective in shielding charge.

Future work

Because the aim of this experiment was to understand the role of PEG on the DNA transformation efficiency, the results could be improved by isolating PEG from the transformation mixture and only exposing that to UV and temperature. However, due to limitations in technical experience and apparatus, exposing the entire mixture was more cost-effective. A further development would be to separate each component to confirm which one is affecting the transformation efficiency.

In future experiments, a more quantitative analysis could be conducted, measuring the molecular weight distribution of PEG after UV exposure using gel permeation chromatography. If the quantity of PEG 3350 degradation could be measured, a more reliable model or explanation could be presented for how PEG contributes to the transformation buffer efficiency on E. coli growth rates. Another method of quantitative analysis for measuring transformation efficiency would be to use gel electrophoresis to run sample DNA fragments before and after treatment with the CRISPR-Cas9 system11. While I was able to put together a plot for low transformation efficiencies as shown in Figure 2 for trial 2, it was impossible to measure growth rates objectively in trial 3 as the transformation efficiencies became much higher.

Appendix

This procedure was taken from The Odin Company’s instructions in the DIY CRISPR Kit (www.the-odin.com/diy-crispr-kit).

Making Plates

  • 6g (in a 15mL tube) of LB Agar Media
  • 250 mL glass bottle
  • 50 mL plastic conical tube (or anything else that can measure water)
  • 300 mL water
  • Microwave
  • Washcloth/hand towel
  • 14 Petri dishes
  • Refrigerator (optional in this step)
  • 6g (in a 15mL tube) of LB Strep/Kan/Arab Agar Media
  1. Take a tube labeled “LB Agar Media” and dump its contents into the 250mL glass bottle.
  2. Using the 50mL conical tube labeled “For Measuring Water”, measure and add 150mL of water to the glass bottle.
  3. Heat the bottle in the microwave for 30 seconds at a time, being careful not to let the bottle boil over. DO NOT SCREW THE LID DOWN TIGHT! (just place it on top and give it a slight turn).
  4. The liquid should look yellow and transparent. This should take about 2-3 minutes total of microwaving. Take the bottle out using a washcloth/hand towel and let it cool. This will take 20-30 minutes.
  5. While the bottle remains somewhat warm, pour the plates. One at a time, remove the lid of 6 plates and pour just enough of the LB agar from the bottle to cover the bottom half of the plate. Put the lid back on.
  6. Let cool for at least one 1 hour before use; this process can be sped up in the fridge. If possible let the plates sit out for a couple hours or overnight to let the condensation evaporate. Then store in the fridge at 4ºC upside down so any condensation doesn’t drip on the plates.
  7. Repeat steps 1-6 using the tube labeled “LB Strep/Kan/Arab Agar Media.” For step 5, instead of pouring 6 plates, pour 8 plates.

Preparing the Transformation Mix

  • Pipette
  • 200µL pipette tips
  • 1 mL transformation buffer 25mMCaCl2, 10% PEG 3350, 5% DMSO
  • 8 microcentrifuge tubes
  • Microcentrifuge tube rack
  • Ruler
  • 1 sterilized contact lens container
  • Rubber band
  • 2 glass test tubes
  • Marker
  • UV Apparatus (made before)
  • Object with surface at least 2” by 2” (used before)
  • Timer/stopwatch
  • Stove
  • Pot
  • Water
  • Candy thermometer
  • Test tube holder
  1. Pipette 100µL of Transformation Mix into a new microcentrifuge tube. Repeat 1 more time. Rubber band a sterilized contact lens container to one end of a ruler. Then, pipette 100µL of Transformation Mix into one end of a the contact lens container. DO NOT PUT THE LIDS ON THE CONTACT LENS CONTAINER. Finally, pipette 100µL of Transformation Mix into a test tube. Repeat one more time. Label one test tube with the number 6 and one with the number 7 if possible.
  2. Number the tubes with Transformation Mix in them 1 and 8. These will be the controls. Then, set aside empty microcentrifuge tubes and label them 2, 3, 4, 5, 6, and 7.
  3. Take the contact lens container (that is attached to the ruler) and gently slide it into the PVC pipe of the UV apparatus. Part of the ruler should be sticking out for easier removal.
  4. Place an object with a surface at least 2” by 2” a few inches away from the PVC pipe opening (for safety reasons).
  5. Turn on the fan, wait until it is completely turned on, then turn on the UV lamp. Leave the UV lamp on for 3 minutes.
  6. Take out the ruler and contact lens container very carefully. Pipette the Transformation Mix from one side of the container into the microcentrifuge tube labeled 2.
  7. Repeat pipetting 100µL of Transformation Mix and steps 3-6 three more times and fill up tubes 3-5. Sterilize the container between each repetition. For #3, keep the mix under the UV light for 6 minutes. For #4, keep the mix under the UV light for 9 minutes. For #5, keep the mix under the UV lamp for 12 minutes
  8. Heat water on the stove to 70˚C. Take the test tube labeled 6 and use a test tube holder to submerge the bottom half of the test tube in the water for 30 seconds. Submerge it for another 30 seconds after taking it out for around 10 seconds.
  9. Heat the water (it can be the same) to 80˚C. Take the test tube labeled 7 and use a test tube holder to submerge the bottom half of the test tube in the water for 30 seconds. Submerge it for another 30 seconds after taking it out for around 10 seconds.
  10. Pipette the Transformation Mix from test tube 6 into the microcentrifuge tube labeled 6. Pipette the Transformation Mix from test tube 7 into the microcentrifuge tube labeled 7.

Making Competent Bacterial Cells for Transformation

  • Nitrile gloves
  • Non-pathogenic E. coli bacteria
  • Inoculation loop(s)
  • 4 LB Agar Media plates (made previously)
  • Transformation mix (made previously)
  1. Put on gloves.
  2. Dip loop into E. coli sample.
  3. Hold loop with bacterial sample parallel to fresh “LB Agar Media” plate.
  4. Gently rub loop across surface spreading bacteria thinly throughout.
  5. Flip loop on other side or even gently drag edges across plate if visible bacteria still needs to be delivered.
  6. The lines on the surface can be seen when the plate is held up to a bright background.
  7. Repeat steps 1-5 for 3 more “LB Agar Media” plates.
  8. Let the plates grow overnight 12-18 hours or until whitish bacteria grow. Avoid placing the plate in areas of high temperature variation like an unheated garage. Consistent and warm temp. locations are preferable. Note: Having fresh bacteria for a transformation greatly increases the likelihood of a successful experiment.
  9. Using an inoculation loop, gently scrape some bacteria off the fresh LB Agar Media plates and mix it into the transformation mix (the 8 tubes we prepared previously). Make sure to either use a new loop for each tube or sterilize it in between.
  10. Mix until any big clumps have disappeared. This might require gently pipetting the mixture up and down. Avoid creating bubbles if possible. The transformation mix should be very cloudy. If it is not, mix in more bacteria until the mixture is opaque.

DNA Transformation and CRISPR Experiment

  • 55µL of 100ng/µL – Cas9 plasmid Kanr
  • Pipette
  • 200µL pipette tips
  • 55µL of 100ng/µL – gRNA plasmid Ampr
  • 55µL of 1mM – Template DNA
  • Fridge
  • Stove
  • Pot
  • Water
  • Candy thermometer
  • Timer/stopwatch
  • Micro centrifuge tubes containing LB broth
  • 8 LB Strep/Kan/Arab Agar Media plates
  1. Find the DNA tube labeled “Cas9 and tracrRNA” and, using a pipette, add 10uL to each of the competent cell mixtures (the tubes labeled 1-8). Change out the pipette tip for a new one.
  2. Find the DNA tube labeled “crRNA” and pipette 10uL to each of the competent cell mixtures. Change pipette tips.
  3. Find the DNA tube labeled “Template DNA” and pipette 10uL to each of the competent cell mixtures.
  4. Incubate the tubes in the fridge (DO NOT FREEZE) for 30 minutes.
  5. Incubate the tubes for 30 seconds in 42˚C (108˚F) water.
  6. Add 1mL of room temperature water to one of the LB media micro centrifuge tubes and shake to dissolve the LB.
  7. Using the pipette, add 200uL of LB media to each of the competent cell mixtures containing DNA.
  8. Incubate the tubes at 37˚C (99˚F) for 2 hour or 4 hours at room temperature. This step allows to bacteria to recover and replicate the DNA and perform the CRISPR engineering process. DON’T skimp on the time, this step is key for the experiment to work.
  9. Take 8 LB Strep/Kan/Arab Agar Media plates out of the fridge and let them warm up to room temperature. Label the Agar Media plate “Control” and label each of the LB Strep/Kan/Arab Agar Media plates from 1-8.
  10. Using the pipette, add 100uL of the CRISPR transformation mixture in tube 1 to the plate labeled 1.
  11. Using an inoculation loop, gently spread the bacteria around the plate and let dry for 10 minutes before putting the lid back on.
  12. Flip the plates upside down to prevent condensation from forming and dripping onto the bacteria.
  13. Repeat steps 10-12 for each of the 7 remaining labeled LB Strep/Kan/Arab Agar Media plates. (Tube 2 corresponds to plate 2, tube 3 corresponds to plate 3, etc.)
  14. Incubate the plates at 37˚C (99˚F) for 16-24 hours or room temperature for 24-48 hours.

Bibliography

[1] Ishwar Das, and Sujeet Kumar Gupta. “Polyethylene glycol degradation by UV irradiation.” Indian Journal of Chemistry, 2005: 1355-1358.

[2] Seongok Han, Chongyoup Kim, Dongsook Kwon. “Thermal/oxidative degradation and stabilization of polyethylene glycol.” Polymer, 1997: 317-323

[3] Emma Haapaniemi, Sandeep Botla, Jenna Persson, Bernhard Schmierer and Jussi Taipale. “CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response.” Nature Medicine. June 11, 2018.

[4]Ekaterina Pak. The Graduate School of Arts and Sciences. 2015. http://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/.

[5] Alice Park, Lon Tweeten, Alexandra Sifferlin. How the Science of CRISPR Can Change Your Genes. June 23 2016, 2016. http://time.com/4377130/crispr-genome-editing/.

[6] Heidi Ledford. CRISPR fixes disease gene in viable human embryos. August 2, 2017. https://www.nature.com/news/crispr-fixes-disease-gene-in-viable-human-embryos-1.22382.

[7] Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, Emmaneulle Charpentier. “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Science, August 2012.

[8] Giedrius Gasiunas, Rodolphe Barrangou, Philippe Horvath, Virginijus Siksnys. “Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.” Proceedings of the National Academy of Sciences, September 2012.

[9] www.the-odin.com/diy-crispr-kit. 2018. http://www.the-odin.com/diy-crispr-kit/.

[10] Higa, Morton Mandel and Akiko. “Calcium-dependent bacteriophage DNA infection.” Journal of Molecular Biology, October 1970: 159-162.

[11] C.T. Chung, Suzanne L. Niemela, Roger H. Miller. “One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution.” Proc. Natl. Acad. Sci. USA, 1989. 2172-2175.

[12] Structural Biochemistry/Nucleic Acid/DNA/DNA structure. 2018. https://en.wikibooks.org/wiki/Structural_Biochemistry/Nucleic_Acid/DNA/DNA_structure.

[13] Bart Gottenbos, Dirk W. Grijpma, Henny C. van der Mei, Jan Feijen, Henk J. Busscher. “Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria.” Journal of Antimicrobial Chemotherapy 48, no. 1 (July 2001): 7–13.

About the Author

My name is Katelyn Chu, and I am currently at junior at St. Francis High School in Mountain View, California. My goal is to engage in scientific research which will later lead me to develop new cancer treatments. Since 2016, I have maintained an online blog (www.katelynchu.wordpress.com) to share my thoughts on STEM related items in the news, like cancer research, gene editing, etc.

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