Team:Heidelberg/Natural Transformation


Natural Transformation





Cloning with Natural Transformation

Background

This part of our project deals with the natural competence of bacteria. The discovery of natural competent bacteria was done in 1990 with Helicobacter pylori which were called Campylobacter pylori. H. pylori is a gram-negative bacteria which can infiltrate the human stomach. From 25 clinically isolated strains, 22 were naturally competent. This resulted in them taking up plasmids with streptomycin resistance spontaneously [1]. For our natural transformation experiments we decided to use the laboratory safety 1 level strain Acinetobacter baylyi ADP1 (A. baylyi). This strain was previously known as Acinetobacter calcoaceticus BD413 or LMD 82.3 and belongs to the group of gram-negative cocci. It is a soil bacterium and can grow on various carbon sources. A. baylyi’s metabolism and genetics are well-known which makes it an attractive chassis for biotechnological applications [2]. A. baylyi has become a model organism for studying costs and benefits of natural transformation as well as type 4 pili (T4P) abundance in microbial strains [3], [4].
The natural transformation mechanisms in gram-positive and gram-negative bacteria are not conclusively clarified [5]. However, it is known that T4P carries an essential role [6]. The T4P and structurally-related protein fiber (T4F) are expressed in the pil, tad and com operons [7], [8]. Generally, T4P carries important functions in microbiota–host interactions, in both commensal and pathogenic bacteria. It is responsible for bacterial motility, biofilm formation, protein secretion, adhesion, DNA uptake and so forth [6]. The gram-positive bacteria Bacillus subtilis 168 (B. subtilis) can be converted into a natural competent stage by overexpression of the ComK gene. With these experiments the influence of T4P for natural transformation was demonstrated as ComK is part of T4P. ComK is also known as the major competence transcription factor engaged in the DNA uptake of Bacillus [9].
Research shows that the natural transformation efficiency is increased by processing the DNA with methods such as Gibson Assembly and others [10]. We got in contact with Dr. Xinglin Jiang, an expert for natural transformation with A. baylyi ADP1, who’s research showed that natural transformation is most effective with multicopy and linear DNA. His idea was to create a rolling circle with the phi29 polymerase but we decided for the overlap extension PCR protocol from [11].
Another technique, to mention in this context, is to create homology flanks for integration of the plasmid DNA into the bacterial chromosomal DNA [12]. Flank tags are sequences which fit to parts of the bacterial chromosome and can be directly integrated into the bacterial genome. This method is widely used to transform B. subtilis because the stability of the uptaken target DNA is higher. General plasmid DNA transformation is rather rarely used for B. subtilis. We received this information from Dr. Ilka Bischofs-Pfeifer who has been working for decades with Bacillus. Unfortunately, this is a non suitable technique for our project because we want the bacteria to take up the transformant DNA as a plasmid and transmit the DNA to other bacteria by conjugation. Bacterial conjugation is performed by the membrane-associated macromolecular machinery called Type IV secretion system and is the best studied horizontal gene transfer machinery [13].

Experimental design and results for overlap extension PCR

One of our milestones in the project was establishing a protocol for our project with natural transformation. Natural transformation is compared to the established transformation protocols like heat shock or electroporation with the model cloning organism Escherichia coli a lot faster and simpler. Both established transformation methods are based on the processing of the bacteria to make them competent to take up DNA during the heat or electro shock. In contrast, for natural transformation the idea is to process the DNA in a way that the bacteria can take up the DNA, circularize it and express new capabilities [5]. This method is widely discussed to become the model transformation method because less harm and stress is imposed on the bacteria for optimal DNA uptake.
There are many different techniques to process the DNA for example Gibson Assembly, overlap extension polymerase chain reaction (PCR) or mutagenesis ligation [12]. We decided to go along with the overlap extension PCR method where we designed overlap extension primers using the protocol from Chun You et al. in order to create a multicopy and linear plasmid in a two step overlap extension PCR [11] (see Fig. 1).

Figure 1: Overlap extension PCR schema. In (1) the linear vector and the insert are represented with the forward and reverse primers. The colors are representative for a certain sequence section. (2) shows the first polymerase chain reaction (PCR) to create the overhangs which should function as primers in the second PCR. (3) is the final overlap extension PCR. In (4) the optimal final PCR product is drawn. Created with BioRender.com

We decided to use the Lvl0_8_Amp/ColE1 plasmid. It was a gift from the part collection of the iGEM Team Marburg to the iGEM Team Heidelberg 2020. The plasmid is short with about 2 kb, carrying ampicillin resistance and the red fluorescence protein mRFP1. In brief, we cut our mRFP1 with the restriction enzyme BsaI to create the linear vector and an insert. Then we designed four primers where we took the first 20 base pairs forward and reverse from the target DNA sequence (e.g. vector) and the overhangs on our primer were the fitting DNA sequence from the second DNA sequence (e.g insert) (see Fig. 2 A).

Figure 2: Overlap extension PCR results Gel and Sequencing. (A) represents the primer sequences fitting to Lvl0_8_Amp/ColE1 after BsaI cut. (B) is the gel with the Lvl0_8_Amp/ColE1 insert and vector PCR and in (C) is the gel with the results of the overlap extension PCR. (D) shows our sequencing results from the insert after the first PCR where in blue are the Primer regions. For the forward and reverse overhang we detected that our pfu polymerase was not able to complete the whole 20 bp overhang. About 8 base pairs were missing on both sides.

Discussion overlap extension PCR

We ran the first PCR with two primer pairs separately for the Amp/CoIE1 backbone and the mRFP 1 insert (see Fig. 2 B). The band in the gel looked promising but we wanted to control the efficiency of our overhangs by sequencing (see Fig. 2 D). We checked the overhangs and found out that about 8 bp were missing from the overhangs of the insert. We also expect this to be the same for the vector sequence because all primer overhangs had a comparable length of around 20 bp. The overhangs are essential for the second PCR because they are supposed to ligate to the complement strand. We only used the PCR products insert and backbone and our polymerase to create a multicopy and linear version of the plasmid (see Fig. 2 C). In our troubleshooting process we changed polymerase from taq to pfu but the results for the second PCR were identical. We should have used the phusion polymerase, as it was recommended in several papers, but the phusion polymerase was not accessible for our team.

Experimental design and results NT in monoculture

After the unsuccessful creation of multicopy and linear DNA strands we discussed again the principle of natural transformation especially for our strain A. baylyi ADP1 to find an alternative strategy. In the beginning we tried to perform natural transformation with the circular plasmid Lvl0_8_Amp/ColE1 and the linearized vector from Lvl0_8_Amp/ColE1 (data not shown). These first trials were unsuccessful. In our halftime discussion we found out that CoIE1 is a non suitable origin of replication (ori) for A. baylyi ADP1 and only works as a negative control. Therefore, the multicopy and linear DNA primers are required to fit to a plasmid with an ori suitable for A. baylyi ADP1. We finally ordered the plasmid from Addgene pBWB162 (BBa_K3963000) designed by Keith Tyo with the suitable A. baylyi ADP1 ori pBAV1k BBa_K3963004. With this plasmid we performed a natural transformation protocol following the instructions from the paper Briggs et al. 2020. The mCherry insert on the plasmid served as a control of successful expression of the Protein in A. baylyi ADP1 (see Fig. 3 B).

Figure 3 Characterization of pBAV1k plasmid in Escherichia coli DH5ɑ and Acinetobacter baylyi ADP1. In (A) is the size definition of the pBAV1k plasmid after preparation from received Escherichia coli DH5ɑ. (B) depicts the naturally transformed A. baylyi ADP1 overnight culture (50 mL) pellet with a clear mCherry expression.

Figure 4: Efficiency of natural transformation in Acinetobacter baylyi ADP1. (A-D) shows the natural transformation plates. (A) is the positive control on a non-selective plate, (B) is the negative control without any plasmid DNA, (C) is the natural transformation with 25 ng of the pBWB162 plasmid and (D) is the natural transformation with 100 ng of the pBWB162 plasmid. The graph (E) shows the efficiency comparison of 25 ng and 100 ng. On the y-axis is defined with the colony forming unit (CFU) and on the x-axis are the different conditions. Additionally, the p values between the two protocols and the amount of DNA was calculated.

Discussion A. baylyi ADP1 NT in monoculture

We compared the efficiency of the natural transformation in monocultures with two protocols and different amounts of DNA. The plasmids used for the natural transformation were the pBWB162 and lacI-PT5-gusA-pBAV1k (Plasmid #30501) designed by Ichiro Matsumura.
We measured a significant difference between the number of colony forming units depending on the amount of injected transformant DNA. The p value between 25 ng to 100 ng is equal to 0.0879 which indicates that the natural transformation success is dependent on the amount of injected DNA. Additionally, we compared the natural competence of A. baylyi in the exponential phase (“log-phase” protocol) by adding DNA in the log-phase versus adding DNA directly to the diluted overnight culture (“overnight” protocol). It can be assumed that the natural competence is higher in the exponential phase but not significant considering the p value for 25 ng with 0.0283 and for 100 ng with 0.0091. The observation that the “log-phase” protocol works better is congruent to the literature [3].

Experimental design and results NT in co-culture

Natural transformation was also performed in co-cultures composed of E. coli DH5ɑ, B. subtilis 168 and A. baylyi ADP1. In order to test the correlations between these three strains we measured the co-culture strains each against each other by fluorescence. We performed the natural transformation according to the monoculture “overnight” protocol except we increased the amount of DNA to 200 ng.

Figure 5: Natural transformation of Acinetobacter baylyi ADP1 in co-culture. (A) shows the counted colonies in a table. The co-culture natural transformation experiment was performed with 100 ng and 200 ng of the pBWB162 plasmid regarding the “overnight” protocol. (B+C) represents the natural transformation co-culture plates with 100 ng plasmid DNA in (B). The successful cloned colonies in (C) with 200 ng of plasmid DNA are depicted with white arrows.

Discussion A. baylyi ADP1 NT in co-culture

The natural transformation was successful in the tripartite of our bacterial strains, although in the co-culture preliminary experiments there was no complete equal growth observed. In our preliminary co-culture experiments we had uniform growth between A. baylyi and E. coli but not between A. baylyi and B. subtilis. B. subtilis had a growth advantage against A. baylyi. One explanation could be that the temperature of 30 °C for the natural transformation, which differed from the 37 °C in the co-culture, had an influence on B. subtilis growth.
Another observation was that the efficiency of our natural transformation was lower because we had to double the amount of DNA before observing positive clones. The mean of colony forming units (CFU) using 100 ng of DNA is 11.5 CFU in monoculture. This was comparable to the 200 ng in co-culture with 11 CFU.

Conclusion for NT with A. baylyi ADP1

The natural competence in A. baylyi ADP1 is well known and was reproducible in our laboratory conditions. The most important aspect is to define a suitable plasmid for this method which is different to the common pET or pUC backbones. Furthermore, the transformation method was working very efficiently after establishing the protocol in our lab. Therefore we would like to discuss this topic further in the following paragraphs.

Acinetobacter baylyi ADP1 as a new cloning model organism

A. baylyi ADP1 appears to show solid and reproducible transformation success. We got in touch with the iGEM Team SCU-China 2021 and discussed the topic of new model cloning organisms. They started a survey to get an overview of how open minded the scientific community is towards new organisms. Additionally they aimed to find out the most important factors to finally switch to another organism. In the following I would like to compare E. coli with A. baylyi and illuminate the advantages and disadvantages of both organisms. A comparison of different tasks needed for transformation for each bacteria and the amount of work they need are compared in the following table:

Escherichia coli DH5ɑ Acinetobacter baylyi ADP1
competent cells 2-3 days, high effort overnight culture, no effort
transformation protocol 1 day, medium effort 5h, low effort
plasmid preparation Kit, low effort homogenizer plus Kit, medium effort
efficiency high N/A
The differences are mainly to be found in the protocol, as the handling can take place on the same broth and under safety level 1 conditions.

In the beginning, E. coli is much more complicated and time-consuming because the cells have to be made competent in a work of 2 days. Then for example the heat shock protocols are also a one-day effort. With A. baylyi you need, if at all, an overnight culture from the glycerol stock, put the DNA on it for 3-5 h and plate the colonies. This is in total a work of one day. Thus, money and time expenditure are very much on the side of A. baylyi. Only the amount of plasmid for A. baylyi would have to be increased, but in monoculture 25 ng are already enough for successful transformation.

With regard to expression, the tide is turning. To get the plasmids from A. baylyi purified one first needs a homogenizer, whereas with E. coli conventional, widely available kits can be used. With A. baylyi, this requires an additional step which costs time and equipment.

Finally, there is another big problem with A. baylyi. Apart from the poor experience and lack of literature, A. baylyi cannot work with the ColE1 ori, therefore the model plasmids pUC, pET and others cannot be used. In our experience this was the biggest issue working with A. baylyi.

The simplest solution would probably be to produce natural competent E. coli, but this is also described in the literature as very difficult [14].

As a conclusion, for industry it is rather unsuitable due to the lack of optimal model plasmids such as pET or pUC and the uncertain efficiency, although rapid cloning in the laboratory could be significantly simplified by A. baylyi.

References

[1] Nedenskov-Sørensen, P., Bukholm, G., & Bøvre, K. (1990). Natural competence for genetic transformation in Campylobacter pylori. The Journal of infectious diseases, 161(2), 365–366. https://doi.org/10.1093/infdis/161.2.365

[2] Kannisto, M., Aho, T., Karp, M., & Santala, V. (2014). Metabolic engineering of Acinetobacter baylyi ADP1 for improved growth on gluconate and glucose. Applied and environmental microbiology, 80(22), 7021–7027. https://doi.org/10.1128/AEM.01837-14

[3] Hülter, N., Sørum, V., Borch-Pedersen, K., Liljegren, M. M., Utnes, A. L., Primicerio, R., Harms, K., & Johnsen, P. J. (2017). Costs and benefits of natural transformation in Acinetobacter baylyi. BMC microbiology, 17(1), 34. https://doi.org/10.1186/s12866-017-0953-2

[4] Ellison, C. K., Dalia, T. N., Klancher, C. A., Shaevitz, J. W., Gitai, Z., & Dalia, A. B. (2021). Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor. Nature communications, 12(1), 3744. https://doi.org/10.1038/s41467-021-24124-6

[5] Smith, H. O., Danner, D. B., & Deich, R. A. (1981). Genetic transformation. Annual review of biochemistry, 50, 41–68. https://doi.org/10.1146/annurev.bi.50.070181.000353

[6] Ligthart, K., Belzer, C., de Vos, W. M., & Tytgat, H. (2020). Bridging Bacteria and the Gut: Functional Aspects of Type IV Pili. Trends in microbiology, 28(5), 340–348. https://doi.org/10.1016/j.tim.2020.02.003

[7] Imam, S., Chen, Z., Roos, D. S., & Pohlschröder, M. (2011). Identification of surprisingly diverse type IV pili, across a broad range of gram-positive bacteria. PloS one, 6(12), e28919. https://doi.org/10.1371/journal.pone.0028919

[8] Piepenbrink K. H. (2019). DNA Uptake by Type IV Filaments. Frontiers in molecular biosciences, 6, 1. https://doi.org/10.3389/fmolb.2019.00001

[9] Nijland, R., Burgess, J. G., Errington, J., & Veening, J. W. (2010). Transformation of environmental Bacillus subtilis isolates by transiently inducing genetic competence. PloS one, 5(3), e9724. https://doi.org/10.1371/journal.pone.0009724

[10] Jiang, X., Palazzotto, E., Wybraniec, E., Munro, L. J., Zhang, H., Kell, D. B., Weber, T., & Lee, S. Y. (2020). Automating Cloning by Natural Transformation. ACS synthetic biology, 9(12), 3228–3235. https://doi.org/10.1021/acssynbio.0c00240

[11] You, C., Zhang, X. Z., & Zhang, Y. H. (2012). Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis. Applied and environmental microbiology, 78(5), 1593–1595. https://doi.org/10.1128/AEM.07105-11

[12] Biggs, B. W., Bedore, S. R., Arvay, E., Huang, S., Subramanian, H., McIntyre, E. A., Duscent-Maitland, C. V., Neidle, E. L., & Tyo, K. (2020). Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1. Nucleic acids research, 48(9), 5169–5182. https://doi.org/10.1093/nar/gkaa167

[13] Huddleston J. R. (2014). Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes. Infection and drug resistance, 7, 167–176. https://doi.org/10.2147/IDR.S48820

[14] Sinha, S., & Redfield, R. J. (2012). Natural DNA uptake by Escherichia coli. PloS one, 7(4), e35620. https://doi.org/10.1371/journal.pone.0035620