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Problem with bacterial transformation with electroporation

Problem with bacterial transformation with electroporation


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I have a problem with a bacterial transformation of a yeast gene that I can not solve.

I isolated yeast DNA and did a PCR to get my product. I am using pCGCUm vector with a GFP construct. I digest both my plasmid and my product with SmaI and ClaI for 4h each. After that I purify the product from a gel and perform a ligation with high efficiency T4 ligase for 30 minutes at 22°C. Then I transform my electrocompetent cells with electroporation (2000V, 5ms), recover it in 0.5ml LB for 2h and plate it on LB-A plates.

First time I only did:

  • insert1
  • insert2
  • Empty vector

and it did not work (empty plates for insert and confluent growth (after 18h) of some weird colonies on EV).

I did the same ligation again and this time I included controls

This is what I did:

  • insert1
  • insert2
  • Empty vector
  • negative control (EK cells + electroporation)
  • positive control (EK cells + undigested vector)

Now I got:

  • insert1 (no colonies)
  • insert2 (big colonies a lot of them)
  • Empty vector (big colonies a lot of them)
  • negative control (EK cells + electroporation) (no colonies)
  • positive control (EK cells + undigested vector) (big colonies a lot of them)

The negative control suggests that my competent cells are ok, at least they are not resistant to AMP.

What confuses me is that even with positive control I get some weird big colonies - if I got nice transformants here I would assume that either digestion of ligation was not successful, but now I doubt that).

Another thing that is weird is that now I got that colonies also on my insert2 plate.

The scan image of big colonies looks like that:

I have a question if anyone has had that kind of colonies? I was thinking of maybe having contaminated cuvettes for electroporation but I had them in 100% EtOH for 3 days and then dried in fume hood for 3 hours before electroporation.


I did try another transformation:

  • again with our electrocompetent cells
  • Cells form other lab
  • chemically competent cells I made

The good thing is that on chemically competent cells there is no weir colonies, however efficiency of transformation was very low, but still, I think it is ok - will confirm it in next day if I really got the right insert.

On the other hand my electroporation still did not clear out. I got colonies on both our cells and cells from other lab, however in much lower amount there. I will try to change the cuvettes and try to use different electroporator as this seems to be the only thing that could make problems - although I am still not convinced about that :)

Thank you for your help and I will report the progress so if somebody else will have that problem again he could maybe benefit from my findings.


Bacterial Transformation Troubleshooting for Beginners

The first time I did a transformation was when I worked with site directed mutagenesis. I cloned a protein sequence into the p15TVL vector, created my mutants (but that&rsquos another story), and was finally ready for the next step: transformation and expression of my desired protein. Little did I know that my enthusiasm would fall off when my first attempts failed (and by first, I mean a month of failure). Because I was a beginner in this technique, my colleagues gave useful suggestions and advice about what could have gone wrong. Below is a simple checklist to use when you don&rsquot see those tiny and precious colonies:


Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains

An important risk factor for acquiring Clostridioides difficile infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Several genetic systems are available to study C. difficile in the laboratory environment, and all rely on stably replicating or segregationally unstable plasmids. Currently, the transfer of plasmids into C. difficile can only be performed by conjugation using Escherichia coli or Bacillus subtilis as conjugal donors. Here we report a method to introduce plasmid DNA into C. difficile using electroporation and test factors that might contribute to higher transformation efficiencies: osmolyte used to stabilize weakened cells, DNA concentration, and recovery time postelectroporation. Depending on the C. difficile strain and plasmid used, this transformation protocol achieves between 20 and 200 colonies per microgram of DNA and is mostly influenced by the recovery time postelectroporation. Based on our findings, we recommend that each strain be tested for the optimum recovery time in each lab.IMPORTANCE Understanding the underlying biology of pathogens is essential to develop novel treatment options. To drive this understanding, genetic tools are essential. In recent years, the genetic toolbox available to Clostridioides difficile researchers has expanded significantly but still requires the conjugal transfer of DNA from a donor strain into C. difficile Here we describe an electroporation-based transformation protocol that was effective at introducing existing genetic tools into different C. difficile strains.

Keywords: Clostridioides difficile electroporation genetics transformation.

Copyright © 2020 Bhattacharjee and Sorg.

Figures

Electroporation of C. difficile R20291…

Electroporation of C. difficile R20291 competent cells grown in sorbitol-containing medium. (A) Electroporated…

Electroporation of C. difficile R20291…

Electroporation of C. difficile R20291 competent cells grown in sucrose-containing medium. (A) Electroporated…

C. difficile competent-cell properties. (A)…

C. difficile competent-cell properties. (A) C. difficile R20291 and CD630 competent cells were…

Transformation of C. difficile strains…

Transformation of C. difficile strains CD630 and JSC10. (A) C. difficile CD630 competent…

Testing the impact of –80°C…

Testing the impact of –80°C storage on eletrocompetence. Competent C. difficile R20291 cells…

Cycling of the anaerobic chamber…

Cycling of the anaerobic chamber air lock does not influence transformation efficiency of…

Competent C. difficile R20291 cells…

Competent C. difficile R20291 cells transformed with different plasmids. pMTL-YN4, pDB29 (a pMTL-YN4…

Confirming the presence of the…

Confirming the presence of the indicated plasmids in C. difficile cells. Thiamphenicol-resistant colonies…


Transformation

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected]

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Videos

The Mechanism of Transformation with Competent Cells

Transformation is the process by which bacteria are made to take up exogenous DNA. The word is derived from Griffith's discovery of a "transforming principle". Learn more about transformation and how it is used in cloning workflows.

Tips for Successful Transformations with NEB ® Competent Cells

Follow these tips to get superior results.

How to Perform a Transformation with NEB ® Competent Cells

Use this protocol for maximum transformation efficiency.

Fast transformation plating with glass beads

In this video, see how transformation plating can be fast and easy using glass beads.


Gene transfer to plants by electroporation: methods and applications

Developing gene transfer technologies enables the genetic manipulation of the living organisms more efficiently. The methods used for gene transfer fall into two main categories natural and artificial transformation. The natural methods include the conjugation, transposition, bacterial transformation as well as phage and retroviral transductions, contain the physical methods whereas the artificial methods can physically alter and transfer genes from one to another organisms’ cell using, for instance, biolistic transformation, micro- and macroinjection, and protoplast fusion etc. The artificial gene transformation can also be conducted through chemical methods which include calcium phosphate-mediated, polyethylene glycol-mediated, DEAE-Dextran, and liposome-mediated transfers. Electrical methods are also artificial ways to transfer genes that can be done by electroporation and electrofusion. Comparatively, among all the above-mentioned methods, electroporation is being widely used owing to its high efficiency and broader applicability. Electroporation is an electrical transformation method by which transient electropores are produced in the cell membranes. Based on the applications, process can be either reversible where electropores in membrane are resealable and cells preserve the vitality or irreversible where membrane is not able to reseal, and cell eventually dies. This problem can be minimized by developing numerical models to iteratively optimize the field homogeneity considering the cell size, shape, number, and electrode positions supplemented by real-time measurements. In modern biotechnology, numerical methods have been used in electrotransformation, electroporation-based inactivation, electroextraction, and electroporative biomass drying. Moreover, current applications of electroporation also point to some other uncovered potentials for various exploitations in future.


Prelab Questions

Discuss the following amongst yourselves. Be ready to share your thoughts with the rest of the class.

  1. Ampicillin is a derivative of the antibiotic penicilliin. It disrupts cell wall formation in bacterial cells which kills the cells. However, our recombinant plasmid contains a gene that provides antibiotic resistance by producing a protein that breaks down ampicillin. Why do we include ampicillin in the test medium?
  2. What will happen if the transformed cells do not grow in the presence of the chemical inducer?
  3. In the experiment, you will add the control and experimental groups of cells onto different media combinations. What do you predict for each condition? Fill in Table 1 by indicating if you predict growth or no growth, and if growth, will there be minimal growth or lots of growth.

Read through the Procedures below and outline the steps, using words and a flowchart in your lab notebook.


Lab Techniques for Molecular Biology- Transformation

2. They allow us to increase the amount of DNA that we have→ if you have a single copy of a particular construct that you're interested in, you can put it into E. coli which will make many copies of the plasmid in a short period of time

3. They have very high fidelity (how much alike the new copy is to the original copy)→ Bacteria make very few mistakes as they are replicating.

4. We can use the host to repair things that are harder to repair in the lab.
ex: dephosphorylate vector so that it will not self ligate. After ligating the vector to our gene of interest, we can put that vector into E. coli and the E. coli can repair the nicks in the DNA caused by the missing phosphate at the 5' ends.

2. This allows us to manipulate the DNA further because we have so much of it. For example, we can hook up gene of interest to GFP or to single sequence that will target it to the ER

3. Shuttle-vectors→ We can produce DNA in a simple system like E. coli and take the plasmid and put it into another organism that the shuttle vector will also work in and grow it in another cell.

-need to have a good post transformation recovery period. This recovery period will allow for proteins that are responsible for the antibiotic resistance (if you are using antibiotic resistance marker in plasmid) in those cells. If the cell isn't given enough time to make those proteins, when exposed to that antibiotic, some cells that have the gene will still die. A good recovery period is typically 60 minutes at 37 degrees C.

-Use cells that are in mid-log phase (instead of cells that are on a plate)→ rapidly growing cells will have a thinner membrane and won't have as cross-linked a cell wall. Log phase cells are much more capable of becoming competent.

-strain of bacteria being used→ DH5α is normally used. K-12 strains and NAB5α were created for the lab (cannot grow well outside lab). Strain and the characteristics of the strain that you use will determine many things like how rapidly it grows and how efficiently does it get through cell wall.

-which divalent cation you use → sometimes you'll use Rb and Ca together which will help decrease the potential damage of too much Ca+.

-heat shock time and temperature→ the amount of time and temperature for heat shock is determined empirically. How the cells are made and the strains you are using will determine these values. For time, you have to follow what has been determined empirically. For temperature, 42 degrees is usually the highest temp you can go. If you go higher, what will typically happen is that the cells will transform but you'll kill them.

-stabilizing the cells on ice (4 degrees C) after heat shock→ if you skip this step, efficiency will decrease a lot. This helps them close the pores in the membrane from the heat shock so that they don't leak their contents into the environment.

-the type of DNA that is being put into the cells→ supercoiled DNA is easy to get into cells. When you are using recombinant DNA, the plasmid has been cut in order to add other pieces to it. This means that you will have relaxed circular DNA, which is less efficient at entering cell. It's harder for this to rapidly enter the pores in the membrane. Linear DNA is poorly transformed because even if it goes into the cell, it is usually degraded from the ends by the endonucleases in the bacterial cell

-size of plasmid→ the smaller the plasmid, the more easier it will go in and the better the transformation. This works like diffusion through the membrane. If plasmid is too large, it will not make it through the pore in time.

The bottom line is that when you are doing transformations in the lab, you want to pay more attention to the competency of the cells that you are using. The competency of the cells tends to be more limiting then the actual amount of DNA that you are putting in.


Transformation

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected]

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Videos

The Mechanism of Transformation with Competent Cells

Transformation is the process by which bacteria are made to take up exogenous DNA. The word is derived from Griffith's discovery of a "transforming principle". Learn more about transformation and how it is used in cloning workflows.

Tips for Successful Transformations with NEB ® Competent Cells

Follow these tips to get superior results.

How to Perform a Transformation with NEB ® Competent Cells

Use this protocol for maximum transformation efficiency.

Fast transformation plating with glass beads

In this video, see how transformation plating can be fast and easy using glass beads.


Gene Pulser Xcell Electroporation System

The Gene Pulser Xcell is a flexible, modular electroporation system for transfecting every cell type from primary, suspension, and difficult-to-transfect cells, including T cells, to bacteria and fungi. The electroporation system uses exponential or square-wave pulses to deliver the pulses optimal for your cell type, and it is built upon the main unit. The CE module contains the low-voltage capacitors required for mammalian cells and plant protoplasts. The PC module contains the resistors needed for high-voltage electroporation. For the highest efficiency while maintaining cell viability, use preset protocols for most common cell types and difficult-to-transfect cells or manually adjust parameters as needed for the best optimized conditions for your cell type.

Full capacity to electroporate both eukaryotic and prokaryotic cells with either exponential-decay or square-wave pulses to optimize conditions that will provide the best efficiency and viability for different cell types.
Delivers low-voltage and high capacitance square-wave pulses to sensitive cells with specified pulse durations and pulse numbers into low-resistance media helping to increase efficiency and viability.
Delivers high-voltage pulses with the ability to control resistance to small volume samples with high resistance, therefore reducing the time constant of an exponential-decay pulse.

Problems with electroporation of Listeria - please, if you have any ideas.. (Jun/09/2011 )

I am trying to transform Listeria monocytogenes EGD with a plasmid for over a 6 months and really need some advice because I am not getting ANY transformed bacteria at all.

I tried several techniques, now I am doing the electroporation according to Monk et al., 2008 (http://aem.asm.org/cgi/reprint/74/13/3921), because they stated that by means of Park and Stewart method EGD is poorly transformed (I did not get anything), and yet do not observe any colonies. Ctrls (bacteria transformed with water instead of plasmid) are growing nicely, though a bit smaller than usually.
Did anybody try this method, or it has some other method that works for him? What are crucial points in transformation of L.m. EGD that could effect it? I am also working with E.coli and it is really easy to transform it, I got a lot of colonies both heat-shock and electroporation method, so this L.m. EGD issues are driving me crazy..

Pretty please, if anybody has any idea..

Thank you very-very-very much!!!

Have you paid any attention to the restriction systems native in the organism? Elimination of restriction sites, or methylation of them with appropriate methylases may solve your problem. Are you certain your resistance cassettes function in L.m.? Have you tried a control transformation with a plasmid known to function in the species?

phage434 on Thu Jun 9 23:48:34 2011 said:

Actually, I did not do that. I cannot find any references regarding restriction systems native in Listeria monocytogenes. Is there any program I can use to check that or how is it done?

phage434 on Thu Jun 9 23:48:34 2011 said:

I am using erythromicin cassette that was already used before in other construct tranformed in L.m.

phage434 on Thu Jun 9 23:48:34 2011 said:

Yes, I have tried it (got it recently as a gift), but still not working, so I am desperate..


Thank you for your kind ideas and help

So, it appears that your first order of business is to get the known working plasmid into your L.m. cells. Until you can do the positive control, there is little sense trying to build and work with new ones that you have no confidence in. I would look very carefully at previous work in transforming L.m. and try to duplicate it. Presumably trying to directly duplicate the experiments of the person who developed the working plasmid is the place to begin.

phage434 on Fri Jul 1 00:08:50 2011 said:

I tried this protocol exactly the same for 5 times with the working plasmid. I know there could be some issues with ratio plasmid/bacteria, but I am measuring the concetration of plasmid, giving exactly 1ug as they described, taking care that the volume does not exceed 10% of volumen of bacteria. Also, there could be some issues with preparation of electrocompetent cells, but they have explained really well how to prepare all the media and buffers (to autoclave or not, to filter-sterilize or not, to adjust pH or not) to use.
The only thing that is not so exact as they say is the electroporator. We have this small Biorad electroporator (Micropulser) that has already programs for different bacteria and yeasts and additionally you can change the voltage (I have set it to 1kV), but you cannot change the resistance(it should be 400 ohms) and the capacity (it should be 25uF) and I could not find anywhere in the manual and at the internet the specification for this matter.
Can that really be such a big factor, so I do not observe ANY of transformed bacteria? :/

Make sure you are using the correct width of cuvette. They come in 4mm, 2mm, and 1 mm width, and you need to use the same as your paper does.

Also, pay careful attention to how they prepare the cells. What phase of growth do they harvest at? How are they washed? I would do a viable colony count on the prepared cells both before and after electroporation by plating out serial dilutions on non-selective plates.

How are you recovering the cells? Do you grow them with non-selective medium for an hour or more, followed by plating on selective plates? What do your co-workers do?

Worst case, can you visit the lab that did these experiments and watch how they do it?

phage434 on Fri Jul 1 12:31:52 2011 said:

I am using the 1mm as they do it in this protocol, so it should not be a problem.

phage434 on Fri Jul 1 12:31:52 2011 said:

The protocol for preparing electrocompetent cells is quiet long and tedious. In short: you have to grow O/N culture in BHI, than dilute this suspension to 10% in BHI medium+ 0.5M sucrose (BHIS). Grow till one stage. Add penicillium. Grow till the OD doubles and then cfg and wash with descending volumes of cold sucrose-glycerol wash buffer (SGWB) for several times. add also in one step lysozime. then cfg and wash again with SGWB and cfg and wash with SGWB. aliquot the suspension in 50uL volumes and store at -80 degrees. Everything on ice all the time.

phage434 on Fri Jul 1 12:31:52 2011 said:

I am always doing a negative control (bacteria+ water) and plating on non-selective plates 3rd and 5th dilution and bacteria grow nicely (the plate is full of bacteria, so I never counted the Nu.). But I will try that to see the exact viable colony count. Thank you for this note )

phage434 on Fri Jul 1 12:31:52 2011 said:

I am recovering them as they said in protocol (actually the protocol is very, very detailed for an article with lots of notes, but it still does not work () : with 1mL room temperature BHIS, statically at 30 degrees in incubator for 1,5h. Do the serial dilutions with BHIS and plate them on selective plates with concentration of selective antibiotic as the people from this other lab, from where I have got the plasmid, have recommended to me.
I am the only person in my lab doing cloning stuff, also the youngest one and I have asked the authors quiet a lot of questions already and I really stick to their advices, but still no result. That is why I asked here, if somebody has some extra advice. Thanks for all your comments and notes. It is really good to go with somebody else through the protocol again and check the crucial points of it.

phage434 on Fri Jul 1 12:31:52 2011 said:

Well, maybe I could be able to arrange that on my own cost during my holidays

Hi, friend. recently i have done this transformation successfully, i hope the method i show below could help you. You should switch the mode to "Time Constant" , then set the voltage and the time to 1000v and 5ms respectively. I was using the 1 mm cuvette and Biorad electroporator. Good luck for you.


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