2007年9月18日星期二
Come back and prepare for my future experiments
I just come back from my vocation. It was a nice journey. I have visited Paris, Berlin and Amsterdam. Now I should sit down and think what I should do next.
2007年9月3日星期一
2007年8月31日星期五
mystery is still mystery
I will go out for a while. I hope I can finish constructing the desired mutants and plasmids before my depature.
I discussed with someone who works about T4 phages. He said that he knows only the injection mechanism about phage infection. Actually, people do not know much about the mechanism about phage infection process as well. He said that the inner membrane receptor has not be found in T4 infection. In addition, porins found to be the receptor of phages does not function as a channel through which phage DNA pass through. Phages use their special ways to circumvent the outer membrane. We also talked about something about porins. He gave me two suggestions: 1. extracting the outer membrane proteins and doing the pull down experiment to see which outer membrane proteins interact with DNA 2. predict outer membrane proteins and screen the candidates by some special programs. Both ways are highly risky and chance is not high. The story about this type of transformation is still a mystery.
I discussed with someone who works about T4 phages. He said that he knows only the injection mechanism about phage infection. Actually, people do not know much about the mechanism about phage infection process as well. He said that the inner membrane receptor has not be found in T4 infection. In addition, porins found to be the receptor of phages does not function as a channel through which phage DNA pass through. Phages use their special ways to circumvent the outer membrane. We also talked about something about porins. He gave me two suggestions: 1. extracting the outer membrane proteins and doing the pull down experiment to see which outer membrane proteins interact with DNA 2. predict outer membrane proteins and screen the candidates by some special programs. Both ways are highly risky and chance is not high. The story about this type of transformation is still a mystery.
2007年8月11日星期六
To survie, everything is possible
No question has been resolved but more questions come out.
1. Is it safe to say that pilQ and comEC are the only channels for DNA passing through the outer membrane and inner membrane now?
For the inner membrane comEC/rec-2 might not be the only channel through which exogenous DNA passes through. My previous experiment clearly shows that transformation did not reduced at all in comEC/ycaI homologue mutant. For the outer membran, pilQ homologue (hofQ and gspD) are not required and so the outer membrane is not exclusive.
2. Stress induce E. coli transformation or nutrient limitation induces this transformation?
Transformation was regulated by rpoS, so transformation should be a response to the general stress. My previous experiment also implicated that crp might play similar role in transformtion. Unfortunately, I did this experiment only one time and after that the mutant was contaminated and always after transformation only white colonies appear on plates and plasmid can not be isolated from these white colonies. So I am not very sure about this result. But I did this experiment with the positive control (Wild type), negative control (rpoS mutant). I used two independent markers to mark the transformant: ampicillin resistance and red fluorescence. Even only with one time result, still I think this should be true. It seems that transformation is regulated both by stress and nutrient limitation. I can just imagine what it might be but it is hard to prove anything since I do not know throug which channels DNA enter into cells.
3. the relation among competence, nutrient limitation and stress. Difficult to distinguish and why we distinguish them?
I repeated Finkel's experiment. Although they lack some essential controls, their conclusion that hofQ is required for DNA used as nutrient is true. I added a control in this experiment, that is let cells grow in minimum culture added glucose to see whether the mutant can eat glucose.
We proposed if hofQ mutant (cloramphnicol resistance) behave the same as wild type cell, when we put the same number of wild type cells and mutant cells in the culture supplemented with glucose, the number of cells on LB plate should be 2 times of that on LB supplemented with cloramphinicol. To our surprise, we get 0 clonies on the plates with chloramphicol and more than 1000 transformants on LB plates (culture was serial diluted). That is wild type cells gain growth advantage even in minimum culture with glucose (they have reported such phenomenon in LB long ago). And such phenomenon was also found in minimum culture added DNA.
Later, I found that hofQ mutant with cat gene cassette grow slower than that of wild type cells in minimum culture with glucose and after I delete the cat gene cassette away, the mutant return back to the normal growth. The mutant without cat cassette can not 'eat' DNA either. Kolter's group has reported that 10-day-old cell can suppress the growth of 1-day-old cell and this phenomenon was regulated by rpoS more than 15 years ago. It is possible that hofQ mutant with cat cassette grow slower (just like 1-day-old cell) and its wild type competitor (like 10-day-old cell) grow quicker and take grow advantage. These results implied that competence homologue gene might be involved in both nutrient acquisition and general stess resposne.
Recent opinions to the evolution of genetic transformation incline to the theory of genetic exchange to gain genetic diversity to cope with stresses in environment, although DNA nutrient theory has also been proposed by some people. In my opinion, the two theory should not conflict, considering that nutrient limitation should be the biggest stress for bacteria in environment.
1. Is it safe to say that pilQ and comEC are the only channels for DNA passing through the outer membrane and inner membrane now?
For the inner membrane comEC/rec-2 might not be the only channel through which exogenous DNA passes through. My previous experiment clearly shows that transformation did not reduced at all in comEC/ycaI homologue mutant. For the outer membran, pilQ homologue (hofQ and gspD) are not required and so the outer membrane is not exclusive.
2. Stress induce E. coli transformation or nutrient limitation induces this transformation?
Transformation was regulated by rpoS, so transformation should be a response to the general stress. My previous experiment also implicated that crp might play similar role in transformtion. Unfortunately, I did this experiment only one time and after that the mutant was contaminated and always after transformation only white colonies appear on plates and plasmid can not be isolated from these white colonies. So I am not very sure about this result. But I did this experiment with the positive control (Wild type), negative control (rpoS mutant). I used two independent markers to mark the transformant: ampicillin resistance and red fluorescence. Even only with one time result, still I think this should be true. It seems that transformation is regulated both by stress and nutrient limitation. I can just imagine what it might be but it is hard to prove anything since I do not know throug which channels DNA enter into cells.
3. the relation among competence, nutrient limitation and stress. Difficult to distinguish and why we distinguish them?
I repeated Finkel's experiment. Although they lack some essential controls, their conclusion that hofQ is required for DNA used as nutrient is true. I added a control in this experiment, that is let cells grow in minimum culture added glucose to see whether the mutant can eat glucose.
We proposed if hofQ mutant (cloramphnicol resistance) behave the same as wild type cell, when we put the same number of wild type cells and mutant cells in the culture supplemented with glucose, the number of cells on LB plate should be 2 times of that on LB supplemented with cloramphinicol. To our surprise, we get 0 clonies on the plates with chloramphicol and more than 1000 transformants on LB plates (culture was serial diluted). That is wild type cells gain growth advantage even in minimum culture with glucose (they have reported such phenomenon in LB long ago). And such phenomenon was also found in minimum culture added DNA.
Later, I found that hofQ mutant with cat gene cassette grow slower than that of wild type cells in minimum culture with glucose and after I delete the cat gene cassette away, the mutant return back to the normal growth. The mutant without cat cassette can not 'eat' DNA either. Kolter's group has reported that 10-day-old cell can suppress the growth of 1-day-old cell and this phenomenon was regulated by rpoS more than 15 years ago. It is possible that hofQ mutant with cat cassette grow slower (just like 1-day-old cell) and its wild type competitor (like 10-day-old cell) grow quicker and take grow advantage. These results implied that competence homologue gene might be involved in both nutrient acquisition and general stess resposne.
Recent opinions to the evolution of genetic transformation incline to the theory of genetic exchange to gain genetic diversity to cope with stresses in environment, although DNA nutrient theory has also been proposed by some people. In my opinion, the two theory should not conflict, considering that nutrient limitation should be the biggest stress for bacteria in environment.
2007年8月1日星期三
plasmid transduction
This morning, I read some referrences about plasmid transduction. Plasmid transduction is using phages extracted from hosts harboring a plasmid transducts a receptor strain and screen the transductants expressing the plasmid marker. This means that the plasmid has recombined to the phage when the phage enter into the host, and that plasmid can be carried into cells on the phage vector.
It might not be surprising from the perspective of bacteriophage infection. But the addition of plasmid can change the infection behavior of phage infection and different plasmids have different abilities of plasmid transduction while the recombination frequency in the host cell keep the same. Whats more, recA is not required for plasmid transduction behavior. All these phenomenon strongly suggest that plasmid in itself affect the infection process.
For natural transformation, phages may know more than us. Maybe phage just makes full use of this mechanism to circumvent the two membrane barrier. Although phages adopt various strategies to pass through the barriers, in the final stage they will have to let their DNA/RNA to cope with the plight. Some lucky phage DNA can be sent to periplasm and face only the inner membrane while some other phage DNA has to face the first membrane as soon as they were released from the phage capsule. Nevertheless, phage DNA (I thinkt there should be no difference between plasmid DNA) must have learned the capability of passing through membranes and capsules and proteins encoded by genes on phage genome just aid DNA entering and enhance the chance of entering and surviving in the host.
Recently, someone argued that RNA virus might be the ancestor of all organism in the earth. Considering that RNA virus is not stable and can be easily degraded, I support the idea that DNA virus should be the ancestor, locating at the root of the evolution tree. It is possible that DNA gradually evolved to DNA virus.
It might not be surprising from the perspective of bacteriophage infection. But the addition of plasmid can change the infection behavior of phage infection and different plasmids have different abilities of plasmid transduction while the recombination frequency in the host cell keep the same. Whats more, recA is not required for plasmid transduction behavior. All these phenomenon strongly suggest that plasmid in itself affect the infection process.
For natural transformation, phages may know more than us. Maybe phage just makes full use of this mechanism to circumvent the two membrane barrier. Although phages adopt various strategies to pass through the barriers, in the final stage they will have to let their DNA/RNA to cope with the plight. Some lucky phage DNA can be sent to periplasm and face only the inner membrane while some other phage DNA has to face the first membrane as soon as they were released from the phage capsule. Nevertheless, phage DNA (I thinkt there should be no difference between plasmid DNA) must have learned the capability of passing through membranes and capsules and proteins encoded by genes on phage genome just aid DNA entering and enhance the chance of entering and surviving in the host.
Recently, someone argued that RNA virus might be the ancestor of all organism in the earth. Considering that RNA virus is not stable and can be easily degraded, I support the idea that DNA virus should be the ancestor, locating at the root of the evolution tree. It is possible that DNA gradually evolved to DNA virus.
2007年7月31日星期二
Searching for new targets for natural transformation of E. coli
Now it is clear that my transformation does not use any competence homologue components. Others have reported another transformation mechanism (in which components of type IV secretion system are required) related to conjugation. We tried to search for these type IV secretion system homologue in E. coli K-12, but nothing was found. To our knowledge, only three form of HGT events: transformation, conjugation and transduction. The candidates for the former two forms of HGT can be excluded. The only linkage that we can refer to is transduction. First of all, we need to find porins which are conserved and required for double strand DNA phage binding or passing through the outer membrane. Then we will test their effect on our cryptic transformation.
Two candidates were screened out——tsx gene and ompA.
The reasons are as following
tsx:
1. involved with the permeation of ribo- and deoxy-nucleosides across the outer membrane of E. coli.
2. serves as a receptor for bacteriophages and colicins.
3. part of its protein has nucleotide affinity.
ompA:
1. non-specific diffusion channel
2. phage receptor
3. mediator of F-factor dependent conjugation (we have the phenotype indicating that cell density is important for transformation and this implies that cell-cell contact might be involved in transformation)
4. rpoS regulated. (rpoS is the only gene which has been identified to be required)
Two candidates were screened out——tsx gene and ompA.
The reasons are as following
tsx:
1. involved with the permeation of ribo- and deoxy-nucleosides across the outer membrane of E. coli.
2. serves as a receptor for bacteriophages and colicins.
3. part of its protein has nucleotide affinity.
ompA:
1. non-specific diffusion channel
2. phage receptor
3. mediator of F-factor dependent conjugation (we have the phenotype indicating that cell density is important for transformation and this implies that cell-cell contact might be involved in transformation)
4. rpoS regulated. (rpoS is the only gene which has been identified to be required)
2007年7月27日星期五
hofQ and gspD are not involved in
My result about gspD mutation has been out. I compared the gspD mutant, hofQ mutant and gspD-hofQ double mutant with the wild type. All can be transformed. I also tested hofQ mutant with Finkel's protocol and the result shows that it is true that hofQ mutant can not use DNA as a nutrient and the wild type can. Then how does DNA enter into cell in my protocol? People here questioned whether the transformation is a genetic event or not. But it is really controlled by rpoS. Another question is why cells do not use hofQ channel to transform but choose another way? It is still quite possible that we do not know the exactly transformation pathway which is under the control of rpoS although the possibility of indirect/mutual effect of rpoS on transformtaion can not be excluded. The main problem is where should I address in the next step since no candidates is available. I am lost again.
2007年7月20日星期五
new mutant construction
Our institution has received a K-12 mutant library recently and I have assayed gspD mutant. It is OK. But I still will transfer this mutant to my strain to ensure the result with my new method, which is quicker than using P1 and even safer for getting mutant than using P1. I do not like using P1 transduction since I made no success at all with this method. So with this mutant library, I should be able to select more candidates even if gspD does not work this time again. I am trying to repeat Finkel's experiment about using DNA as a nutrient to see whether hofQ actually work in 'eating DNA' in my strain. My recent data support DNA can be used as a nutrient in ZK126. But my strain did not grow in the minimum culture even supplemented with glucose. This might be resulted of lacking of some amino acides. After adding these amino acides, I will see whether hofQ is really necessary for eating DNA.
2007年7月12日星期四
Game over and play again
Ok, I should forget about the traditional pathway and try to find other ways. We should return back to the initial question --how does DNA pass throught the first membrane? There are two pilQ homologue in E. coli, hofQ and gspD. HofQ has been reported to be required for DNA as a nutrient, however, it is not the channel for our transformation. I will test the second candidate gspD and the primers should be on the way. If it is true, this might tell us the story of the criptic regulon gsp. If not, we will be blind again.
I discussed with my mentor about exploring real chromosomal transformation. He warned me that innumerous failures were on this direction for years before. We do not know whether sxy gene, which is required for natural transformation in H. influenzae and V. cholerae, express in E. coli and if so under which condition it can be turned on. In addition, in V. cholerae, except for sxy, HapR is required for the V. cholerae transformation. We do not know whether similar or additional regulators are required for turnning on competent genes in E. coli. We have evidences showing that rpoS is required for our transformation. HapR is under the regulation of rpoS in V. cholerae. However, hapR homologue was not found in E. coli genome.
I discussed with my mentor about exploring real chromosomal transformation. He warned me that innumerous failures were on this direction for years before. We do not know whether sxy gene, which is required for natural transformation in H. influenzae and V. cholerae, express in E. coli and if so under which condition it can be turned on. In addition, in V. cholerae, except for sxy, HapR is required for the V. cholerae transformation. We do not know whether similar or additional regulators are required for turnning on competent genes in E. coli. We have evidences showing that rpoS is required for our transformation. HapR is under the regulation of rpoS in V. cholerae. However, hapR homologue was not found in E. coli genome.
2007年7月7日星期六
who is the winner in sex evolution? ssDNA or dsDNA?
In prokaryotic cells, ssDNA translocation has widely been adopted in natural transformation and it seems that this type of transformation take an advantage in the evolution of prokaryotic kingdom. In contrast, oocytes receive dsDNA from germ cells and keep it as the double strand form before recombination in the eukaryotic realm. From the perspective of the whole biological evolution, it seems that DNA enter cells with dsDAN form is preferred. The question is, under natural conditions, why prokaryotic cell prefers utilizing ssDNA and degrading the other strand before recombination to making the use of both strands to keep the fidelity of recombination. If we support the idea that eukaryotic cells are evoluted from prokaryotic cells, the prokaryotic transformation should be a inferior form of sex left by the evolution process and there should be some reminiscence of transition this inferior sex behavior to the more advanced one. Considering the importance of sex in proliferation of eukaryotic species, trying to find the reminiscence would reveal some fundamental mechanisms of sex in eukaryotic kingdom. The first step to find the reminiscence is trying to confirm that dsDNA is able to enter into prokaryotic cells and keep its sexual biological activity under natural condition. Then we need to prove that the uptake dsDNA is able to recombinate with the genome DNA of the recipient cell. We know that millions of sperm cells (we can consider these are the donor DNA) are required for a successful impregnation event. If it is true that dsDNA recombination in prokaryotic cells is resemble to impregnation, the chance of successful reombination between dsDNA and genome might also not be very high. However, these accidents pioneered a new biology realm which comprises predominant species in the earth nowadays.
(Note: although no direct evidence is available for proving the dsDNA translocation, some evidences strongly support the existance of dsDNA translocation and the competition between dsDNA transfomration and ssDNA transformation under natural or non-physiological conditions. However, it lacks evidences to prove recombination between dsDNA and chromosomal DNA events under natural conditions)
(Note: although no direct evidence is available for proving the dsDNA translocation, some evidences strongly support the existance of dsDNA translocation and the competition between dsDNA transfomration and ssDNA transformation under natural or non-physiological conditions. However, it lacks evidences to prove recombination between dsDNA and chromosomal DNA events under natural conditions)
2007年7月5日星期四
The implication of non-classical transformation and how to address
I read a recently published paper about genome transplantation. In their report, genome transformation is applicable although with low frequency. They reported that only circular genome can be successfully transformed. But the mechanism of this type of transplantation is completely unkown. I was surprised to found that Dr. Smith is within the authors because I did not find his publications after twentith century. He has done perfect work in transformation of H. influenzae and his publications are thoughtful and insightful.
One teacher in our lab remind me one thing: what is artificial transformation? If there is no way to let DNA enter cell, there is no way for DNA entering. If there is a window there, the outer stress might make the window become a door. My question is that whether the mechanism of plasmid transformation is just the the window of so called 'artificial transformation'. People make this widow become bigger and then the transformation efficiency enhanced so the window was reformed to the door. Natural plasmid transformation has been widely found (here I mean the plasmid without DUS, if the plasmid contain DUS the transformation form changed to chromosomal transformation). Both H. influenzae and N. gonorrhoeae have such kind of transformation. Although it is not very clear whether this type of transformation exists in B. subtilis, the existance of recA-independent plasmid transformation implied that this type of transformation might also apply to gram positive bacteria. However, it is difficult to study this type of transformation at this moment because we do not know whether genes are involved in the regulation (since all competence genes are not required and we might not be able to use genetic methods). In addition, the low transformation frequency make it difficult to observe single competent cells.
Nevertheless, one can completely change the plasmid transformation to chromosomal transformation if the DUS sequence is found. The question is whether DUS sequence really exists in E. coli. Although there are reports which shows negative evidences from bioinformatics, still it is hard to say whether DUS sequence is really abscent in E. coli genome or whether it will appear in another form. In H. pylori, it is not known that whether special DNA uptake sequence is required for natural transformation and the mechanism of transformation in H. pylori is quite novel. We do not know whether the chromosomal transformation of E. coli make use of another novel transformation mechanism.
The following work seems to be a big project. But we need to design several simple experiments to reveal some important aspects and impact firstly. I will discuss with my mentor in detail about what and how I should address to next week.
One teacher in our lab remind me one thing: what is artificial transformation? If there is no way to let DNA enter cell, there is no way for DNA entering. If there is a window there, the outer stress might make the window become a door. My question is that whether the mechanism of plasmid transformation is just the the window of so called 'artificial transformation'. People make this widow become bigger and then the transformation efficiency enhanced so the window was reformed to the door. Natural plasmid transformation has been widely found (here I mean the plasmid without DUS, if the plasmid contain DUS the transformation form changed to chromosomal transformation). Both H. influenzae and N. gonorrhoeae have such kind of transformation. Although it is not very clear whether this type of transformation exists in B. subtilis, the existance of recA-independent plasmid transformation implied that this type of transformation might also apply to gram positive bacteria. However, it is difficult to study this type of transformation at this moment because we do not know whether genes are involved in the regulation (since all competence genes are not required and we might not be able to use genetic methods). In addition, the low transformation frequency make it difficult to observe single competent cells.
Nevertheless, one can completely change the plasmid transformation to chromosomal transformation if the DUS sequence is found. The question is whether DUS sequence really exists in E. coli. Although there are reports which shows negative evidences from bioinformatics, still it is hard to say whether DUS sequence is really abscent in E. coli genome or whether it will appear in another form. In H. pylori, it is not known that whether special DNA uptake sequence is required for natural transformation and the mechanism of transformation in H. pylori is quite novel. We do not know whether the chromosomal transformation of E. coli make use of another novel transformation mechanism.
The following work seems to be a big project. But we need to design several simple experiments to reveal some important aspects and impact firstly. I will discuss with my mentor in detail about what and how I should address to next week.
2007年7月2日星期一
Come back
I enjoyed a good weekend. Together with my friends, we went to Nice. It is quite a beautiful city and the sean is charming there. After that, we went to see the lavande and return back late in the night. It is quite interesting but I am a little tired now. I need to restart my work again and choose a point to dig out the mystical veil.
2007年6月22日星期五
All fall into black again
I discussed with my mentors about my recent results. They should be right. All my recent results tell me only a negative result -- there is not any relation between the transformation and ycaI and dprA mutation.
Firstly, the effect of ampicillin should be just a result of lottery. If I continue increase the repeats of my experiments, the SD will become larger and larger and the difference between the mutants and their parents will become smaller and smaller. That is, actually neither increase nor decrease when I mutate the two genes.
Secondly, the reason for why cells pretreated with antibiotics produce transformants quicker should be a reason of metabolic reason. Because antibiotics kill non-transformants and make transformants grow better. All the results has little relation to transformation mechanism. And I was cheated by the superficial phenomenon.
Nonetheless, these results absolutely demonstrated that ycaI and dprA are not necessary to this type of transformation. We still know nothing about the mechanism of natural transformation in E. coli.
This is a bad result. But I have to face to the fact.
Firstly, the effect of ampicillin should be just a result of lottery. If I continue increase the repeats of my experiments, the SD will become larger and larger and the difference between the mutants and their parents will become smaller and smaller. That is, actually neither increase nor decrease when I mutate the two genes.
Secondly, the reason for why cells pretreated with antibiotics produce transformants quicker should be a reason of metabolic reason. Because antibiotics kill non-transformants and make transformants grow better. All the results has little relation to transformation mechanism. And I was cheated by the superficial phenomenon.
Nonetheless, these results absolutely demonstrated that ycaI and dprA are not necessary to this type of transformation. We still know nothing about the mechanism of natural transformation in E. coli.
This is a bad result. But I have to face to the fact.
2007年6月20日星期三
The conclusion drawn from my older posts
I will discuss with my mentors about my recent results. After I prepare the PPT to be shown tomorrow, I am aware of the following points.
1. I have no need to add DNase in the replica plate to confirm that transformation occur before replica. Because the the replica plate with high concentration of ampicillin and the replica with high concentration of spectinomycin has the same layout when cells pretreated with antibiotics. Transformation must have happened before replica. Otherwise, one transformant can not appear in two different places. This result confirmed that transformation process should finished 10 h after plating.
2. The discrepancy of the layout in the replica containing spectinomycin and ampicillin in the wild type cells might imply that transformation in the wild type can still take place after replcia. However, in the mutant this ability was lost. But the layout discrepancy between replica-Amp and replica-Spt does not appear stably. It is repeatable but not always like this. Nevertheless, if the latter one is really chromosomal transformation. The chromosomal marker should be able to transform in this way. Nevertheless, I will try this simple experiment recently to see whether it is the real transformation.
3. similar effect of low concentration of ampicillin and spectinomycin on transformation has been confirmed recently. The data is better than that I get previously from the wild type. However, several conflicts come out. I can not explain which one is true which one is false. I will discuss them tomorrow.
1. I have no need to add DNase in the replica plate to confirm that transformation occur before replica. Because the the replica plate with high concentration of ampicillin and the replica with high concentration of spectinomycin has the same layout when cells pretreated with antibiotics. Transformation must have happened before replica. Otherwise, one transformant can not appear in two different places. This result confirmed that transformation process should finished 10 h after plating.
2. The discrepancy of the layout in the replica containing spectinomycin and ampicillin in the wild type cells might imply that transformation in the wild type can still take place after replcia. However, in the mutant this ability was lost. But the layout discrepancy between replica-Amp and replica-Spt does not appear stably. It is repeatable but not always like this. Nevertheless, if the latter one is really chromosomal transformation. The chromosomal marker should be able to transform in this way. Nevertheless, I will try this simple experiment recently to see whether it is the real transformation.
3. similar effect of low concentration of ampicillin and spectinomycin on transformation has been confirmed recently. The data is better than that I get previously from the wild type. However, several conflicts come out. I can not explain which one is true which one is false. I will discuss them tomorrow.
2007年6月16日星期六
New finding with some bugs
I have done 10 independent experiments to show that transformation frequency in ycaI mutant is about 5 folds higher than that of its wild type when Ampicillin concentration is 5 microgram per mL. Why my recent data shows that such effect disappeared, and transformation frequency of ycaI mutant is extremely low? Maybe the plate with which I picked up single colony to culutre is too old, I might have to restreak my plates with cells from the -70 refrigerator.
Nevertheless, after several repeats with large amount work (I am very tired this week because I have to go back home after 23:00 in these days), several points are clear. And I know some of the reasons why transformants can not be observed when protein inhibitor was used as the selection marker. Transformants appear much slower when protein inhibitor was used in transformation. The replica was done 10 h later than spreading and transformants can be observed when ampicillin was used as the selection marker 12 h later than replica. Few transformants can be observed when protein synthesis inhibitor was used as selection marker at this time. The latter type of transformants appear 12 h later than replica. That is, in all about 36 h is required for the formation of visible transformants. Transformants can be obviously observed until 48 h after plating or even later. This is quite a long time. In my previous experiments, I should have thrown plates away before the transformants visible. For transformants appeared on ampicillin plates, no such delay can be observed. All transformants appeared after 12 h later than replica. I also noticed that the 48 later appeared transformants has completely different layout comparing with other samples. Only the wild type behave like this. YcaI mutant has no such phenomenon. Both transformants appear 48 h later and the layout of transformants did not appear in YcaI mutant and samples pretreated with antibiotic (neither ampicillin pretreatment and spectinomycin pretreatment). With these information accumulated, it is quite possible that transformants appeared 48 h later should be the chromosomal transformants.
Strangely, MG1655 wild type did not produce transformants after 48 h. I need to redo this experiment and compare it with MG1655::dprA. Or I can do experiment in RR1, because recently I am very lucky in mutant construction. For more than one month, I failed to get dprA mutant in RR1. But luckily, I get it this week, and hofQ as well. Next week, I will have a lot of work to do. Firstly I need to isolate a large amount of DNA for my following experiments. But first of all, I need some rests......
Nevertheless, after several repeats with large amount work (I am very tired this week because I have to go back home after 23:00 in these days), several points are clear. And I know some of the reasons why transformants can not be observed when protein inhibitor was used as the selection marker. Transformants appear much slower when protein inhibitor was used in transformation. The replica was done 10 h later than spreading and transformants can be observed when ampicillin was used as the selection marker 12 h later than replica. Few transformants can be observed when protein synthesis inhibitor was used as selection marker at this time. The latter type of transformants appear 12 h later than replica. That is, in all about 36 h is required for the formation of visible transformants. Transformants can be obviously observed until 48 h after plating or even later. This is quite a long time. In my previous experiments, I should have thrown plates away before the transformants visible. For transformants appeared on ampicillin plates, no such delay can be observed. All transformants appeared after 12 h later than replica. I also noticed that the 48 later appeared transformants has completely different layout comparing with other samples. Only the wild type behave like this. YcaI mutant has no such phenomenon. Both transformants appear 48 h later and the layout of transformants did not appear in YcaI mutant and samples pretreated with antibiotic (neither ampicillin pretreatment and spectinomycin pretreatment). With these information accumulated, it is quite possible that transformants appeared 48 h later should be the chromosomal transformants.
Strangely, MG1655 wild type did not produce transformants after 48 h. I need to redo this experiment and compare it with MG1655::dprA. Or I can do experiment in RR1, because recently I am very lucky in mutant construction. For more than one month, I failed to get dprA mutant in RR1. But luckily, I get it this week, and hofQ as well. Next week, I will have a lot of work to do. Firstly I need to isolate a large amount of DNA for my following experiments. But first of all, I need some rests......
2007年6月14日星期四
Similar phenomenon in dprA mutant
For days, I tried to transform MG1655 with my previous natural transformation protocol. It did not work very well at that time. I constructed MG1655::dprA mutant and tested it with my modified transformation protocol. It seems work. Similar phenomenon in ycaI and its wild type counterpart was observed. No transformants can be observed in the wild type MG1655 after 12 h of replicate the lawn from LA to LA containing spectinomycin. However, transformants can be observed in the MG1655::dprA mutant sample. This is in consistant with the phenomenon which I observed in YcaI mutant. I have to wait until tomorrow to see the layout of the wildtype and compare it with the dprA mutant. Also, I observed the same phenomenon that cells pretreated by antibiotic appear earlier and grow quicker. The transformation induction effect of antibiotics also exist in MG1655 and MG1655::dprA.
Although the data seem good, I was troubled by some personal affairs. The fund for my phD study is available for only one year. When I tried to apply the fund for another year, so few programs about this field are available for my current identity (neither the beginner nor the postponer) while many programs supporting cancer research can be found. Without financial support, my research for the next year will get slow again. Currently, I need to spend some time to try to find an appropriate program to continue my study.
Although the data seem good, I was troubled by some personal affairs. The fund for my phD study is available for only one year. When I tried to apply the fund for another year, so few programs about this field are available for my current identity (neither the beginner nor the postponer) while many programs supporting cancer research can be found. Without financial support, my research for the next year will get slow again. Currently, I need to spend some time to try to find an appropriate program to continue my study.
2007年6月11日星期一
Two Evidences Proving the Activity of YcaI (com2 homologue)
Firstly I provide the two observations which strongly suggest that ycaI dependent transformation actually also participate in our transformation system.
1. 24 h after replica, only the wild type without antibiotic stimulation can still produce transformants. The ycaI mutant, the wild type and the ycaI mutant pretreated with low concentration of antibiotics gives few (most are 0) transformants at this period. It seems that antibiotics induce transformation occurring earlier. This might be true for the ycaI-independent pathway, but another evidence reveal that the two mechanism seems not be the same.
2. When I do the replica experiment, I replicate the lawn from LA plates to two plates, one contains high concentration of Ampicillin and the other contains high concentration of spectinomycin. In ycaI mutant, comparing the LA-Amp plate and LA-Spc plate, the layouts of transformants are almost the same. However, for the wild type, the layouts of transformants on LA-Amp and LA-Spc are completely different.
How to explain this phenomenon. Remember my last post, since transformation occurrs only on plates we can observe transformants in situ. Two possibilities might be able to explain this phenomenon: 1. transformants moved. 2. the two transformantion mechanism are different, so the transformants are derived from different types of competent cells. So they are intrincically different. If first possibility is true, how cells move? Does this means that type IV pili is active at this moment and largely enhanced the mobility of transformants cells? One thing is clear that ycaI-dependent transformation behavior is slower than that of ycaI-independent transformation behavior (the first evidence I posted here). If the second possibility is true, that is the transformants observed on ampicillin plate and spectinomycin plate derived from different cells. Why one type of cells can grow on ampicillin plate and the other grow only on spectinomycin? Since they have finished transformation process and both of the amp resistance gene and spc resistance genes have entered into cells, they should be able to resistant to both of the two type of transformants. The situation is similar to the paper published more than 20 years ago. The author said that they can not explain the phenomenon, that transformation of ampicillin marker in ycaI mutant with a plasmid containing USS is much lower than transformation of ampicillin marker in the wild type cell with the same plasmid without USS. But for nov resistance marker, the former for of transformation was much stronger than the latter one. Both of my results and the author's support that once cells decide to adpot one type of transformation, the other type is closed.
1. 24 h after replica, only the wild type without antibiotic stimulation can still produce transformants. The ycaI mutant, the wild type and the ycaI mutant pretreated with low concentration of antibiotics gives few (most are 0) transformants at this period. It seems that antibiotics induce transformation occurring earlier. This might be true for the ycaI-independent pathway, but another evidence reveal that the two mechanism seems not be the same.
2. When I do the replica experiment, I replicate the lawn from LA plates to two plates, one contains high concentration of Ampicillin and the other contains high concentration of spectinomycin. In ycaI mutant, comparing the LA-Amp plate and LA-Spc plate, the layouts of transformants are almost the same. However, for the wild type, the layouts of transformants on LA-Amp and LA-Spc are completely different.
How to explain this phenomenon. Remember my last post, since transformation occurrs only on plates we can observe transformants in situ. Two possibilities might be able to explain this phenomenon: 1. transformants moved. 2. the two transformantion mechanism are different, so the transformants are derived from different types of competent cells. So they are intrincically different. If first possibility is true, how cells move? Does this means that type IV pili is active at this moment and largely enhanced the mobility of transformants cells? One thing is clear that ycaI-dependent transformation behavior is slower than that of ycaI-independent transformation behavior (the first evidence I posted here). If the second possibility is true, that is the transformants observed on ampicillin plate and spectinomycin plate derived from different cells. Why one type of cells can grow on ampicillin plate and the other grow only on spectinomycin? Since they have finished transformation process and both of the amp resistance gene and spc resistance genes have entered into cells, they should be able to resistant to both of the two type of transformants. The situation is similar to the paper published more than 20 years ago. The author said that they can not explain the phenomenon, that transformation of ampicillin marker in ycaI mutant with a plasmid containing USS is much lower than transformation of ampicillin marker in the wild type cell with the same plasmid without USS. But for nov resistance marker, the former for of transformation was much stronger than the latter one. Both of my results and the author's support that once cells decide to adpot one type of transformation, the other type is closed.
2007年6月8日星期五
Dialectics—The Potential of Application Transformation on Solid Surface Technique
Some of my words might be illogical because of my poor writting. Of course, researchers concern about the factor - time. What I want to express is that few people concern on the time when transformants begin to accumulate on plates because they think that transformation occur only in liquid culture in transformation study. It is possible that this is true for other bacteria. But in E. coli, I have to keep this in mind that transformation occurs only on plates. To evaluate transformability, the number of transformants (transformation frequency and transformation efficiency) should not be the only index. There should be some other aspects which are ignored by others but we should take care . Goodgal published his paper in Nature about 35 years ago and mentioned that different types of H. influenzae mutants with transformation deficiency has different irregular shapes. However, this is just the property of the donor strain not be transfomed. Dubnau's group finds that many genes regulated by comK do not function in competence and DNA uptake and proposed K-state. But no little functions were found in these genes when the competence occur. Claverys' group claims that fratricide accompanies with competence and named X-state. This shows the effect of competence on other cells. No referrences can be found about the properties of transformants in themselves other than the transformation behavior. Is it possible that the transformants which have absorbed exogenous DNA has other abnormal phynotypes (e.g. shape, size) just during transformation? Can they be the indexs for transformation ability evaluation and classification. The transformation process might be dynamic and has an integral impact on the recipients.
We should not just think the black side of transformation on plates (not easy to monitor, not easy to be observed under microscopy and so it is difficult to assay with fluorescence or beta-gal). Nevertheless, we can see the in situ landscape: diversity of transformants just at the position where they were transformed. In liquid cultures, we have no way to discriminate the diversity, however, on plates we can open a window to see its original appearance. The techniques in developing transformation on solid surface could provide much transformation information unconscious before and in turn it calls for other novel techniques to overcome its shortcomings in that not be able to be assayed in liquid culture.
We should not just think the black side of transformation on plates (not easy to monitor, not easy to be observed under microscopy and so it is difficult to assay with fluorescence or beta-gal). Nevertheless, we can see the in situ landscape: diversity of transformants just at the position where they were transformed. In liquid cultures, we have no way to discriminate the diversity, however, on plates we can open a window to see its original appearance. The techniques in developing transformation on solid surface could provide much transformation information unconscious before and in turn it calls for other novel techniques to overcome its shortcomings in that not be able to be assayed in liquid culture.
2007年6月7日星期四
The picture getting clearer
The effect of rec-2 homologue on transformation can not be observed directly because some other factors influenced the results. Often, we try to get the final result, we test all kinds of conditions and factors, but we forget another parameter--the time. In my opinion, gene regulation should not be statical during cell life. On the contrary, it should be variable with the growth. When I counting my transformants, I was always bothered by transformants which occur after I had already counted. I have never thought of the effect of time on transformation until recently. I compared transformants at different time incubation on plates and found that the response to incubation time is so different between rec-2 homologue mutant and the wild type. Especially between 24 h to 36 h incubation on selective plates. Wild type cells that without stimulation by the first round of low concentration of antibiotics behaves weak in the first 12 h but very strong at the last 12 h. 24 h after incubation on plates, ycaI mutant stoped producing transformants, but wild type cell produced a lot of transformants. This strongly suggested that ycaI gene faciliated transformation after 24 h of incubation on plates. Although there are many other important conclusions can be drawn from my recent results, this should be the most important one. Before making any conclusions, I have to repeat all my experiments with more controls to eliminate other possibilities.
2007年6月5日星期二
Modification on Transformation protocol opens new windows
I modified my transformation protocol by chance. Just as I found the transformation protocol by a tube to be thrown away. This time it is the plate which seems of no use tells me something really interesting. Firstly, when the stimulating ampicillin is about 5 microgram per mililiter (actually it should be 2 microgram per mililiter with particularly new). Ampicillin degrades very quick. Even after several days, you will find that one has to add two volume of that used previously for suppressing cells at the threshold concentration. It is quite complicate to explain all my data that I recently get and I do not have much time to write at this moment because I have experiments to do and I am hurry to go back for my meal. I present some of my conclusions here. Ampicillin do affect transformation. But it is difficult to detect when you just use the wild type cell and the rec-2 homologue (I use rec-2 here because many people might not be familiar with ycaI in E. coli, I hope this can be changed after my results are published). When the concentration of ampicillin is fixed at 5, the difference between wild type and the rec-2 homologue mutant is quite obvious (more than 6-fold difference without overlapping in the SD). The result can be covered if I try to test it is ampicillin or the ycaI lead to this difference. Importantly, it is ycaI mutant which has higher transformation frequency. I tested transformation with a plasmid conferring both amp and spc resistant, the second selection marker of an antibiotic which inhibits protein synthesis. Similar results were obteined since no difference if the second selection plates containing ampicillin or spectinomycine. This further confirm that ampicillin should has an effect to enlarge the difference between wild type and rec-2 mutant. This implied rec-2 homologue in E. coli is active although with an adversal effect. Often the friend and the enemy come together. I have many words to say, but I have no time now. Talk later.
2007年5月29日星期二
The risk of disturbing a balance-Non-specific DNA attack
Although the result is good and probablely the result can tell me many things. Still several questions are unresolved. Firstly, even without antibiotic transformants can be detected. Secondly, cat gene can also be transformed in the similar way.
I observed another important phenomenon that transformants prefer grow near low antibiotic concentration region to the high region. This might be not surprise since all of us prefer some cozy places and bacteria as well. However, rec-2 mutant transformants does not like low antibiotic regions. Instead, most transformants appear at middle concentration and even a little high concentration place. What does it tell us? For application, it means that if we design a drug targeting on Rec-2/ComEC protein we will activate another transformation pathway and make things worse. I believe this type of transformation did not confine to plasmid. But indeed, it is hard to prove that this type of transformation also occur in genome. Because if it is true, then the type of recombination should not be homologous transformation. Since non-specific DNA has no homologous regions for recombination. Also, the host cell will employ diverse ways to protect the attack of exogenous DNA. Otherwise, this species will be 'eaten' by another species whoes DNA is highly infectious. Nontheless, non-specific DNA still have special skills to penetrate barriers because we can see their trace through genome comparison.
I have another interesting phenomenon as well. But I have to time to write because I have to do my experiments now.
I observed another important phenomenon that transformants prefer grow near low antibiotic concentration region to the high region. This might be not surprise since all of us prefer some cozy places and bacteria as well. However, rec-2 mutant transformants does not like low antibiotic regions. Instead, most transformants appear at middle concentration and even a little high concentration place. What does it tell us? For application, it means that if we design a drug targeting on Rec-2/ComEC protein we will activate another transformation pathway and make things worse. I believe this type of transformation did not confine to plasmid. But indeed, it is hard to prove that this type of transformation also occur in genome. Because if it is true, then the type of recombination should not be homologous transformation. Since non-specific DNA has no homologous regions for recombination. Also, the host cell will employ diverse ways to protect the attack of exogenous DNA. Otherwise, this species will be 'eaten' by another species whoes DNA is highly infectious. Nontheless, non-specific DNA still have special skills to penetrate barriers because we can see their trace through genome comparison.
I have another interesting phenomenon as well. But I have to time to write because I have to do my experiments now.
2007年5月27日星期日
Antibiotic also induces another transformation pathway
My experiment shows that antibiotic can induce the cryptic transformation. Importantly, transformation was not completely dependent on antibiotic because even antibiotic was abscent, still a few transformants were observed. This indicate the the ability of transformation is inherent and antibiotic just induce (not trigger) transformation. The addition of antibiotic can induce transformation in the rec-2 mutant about 20-fold. I was shocked. A paper published in Science reported that antibiotic can induce natural transformation in S. pneumonia. My data indicate that antibiotic can also induce the non-traditional transformation as well. Still, I do not know how antibiotic induce this cryptic transformation since we know little about it. This is a completely new area waiting for further investigation and the implication of this study is immense (read my post yesterday).
2007年5月26日星期六
Butterfly effect in HGT
Although many people know the butterfly effect, the wave of butterfly wing at one corner of earth can cause tsunami in another place, many scientific researchers forget this story in their actual study unconsciously. Afraid of inexorable critique, only events with high possibility were concerned and events with low possibility and not easily detectable were neglected or were considered to be ignorable 'background'. Take recent research in genetic transformation for example. Admittedly, transformation with homologous DNA was much easier than that with heterologous DNA. As one can get more obvious phenotype in homologous transformation and the evidence with large magnitude difference in their data presented make the arguer standing on a better ground for defencing their theory. However, nature is not just arguing for theory. Like fatal diseases appear out of our conciousless but develop irreversible, many events take place unappreciable but with immense impact in the later phase. Although we get much information about the later events of tranfsormation, our recent transformation mechanism study is just confined to the tsunami and the butterfly was almost completely forgotten.
From the evolution perspective, homologous tranformation makes significant contribution to a special species, but make little impact to the whole ecosystem. The genetic communication between the same species is common to all over the eukaryotic and prokaryotic kindom. So, it is not surprise to discover natural transformation among bacteria of the same species with their own chromosomal DNA. However, this can not explain high frequency event of HGT among different bacterial species providing the information from comparing known genomes. Neitheir conjugation can explain this because gene communication in this form occurs only in conjugative plasmid, another special form of DNA with non-comprehensive impact. Butterfly event might edify us to find the right way to this important issue. The first step of explaning high HGT among different species is to prove that heterogenous DNA is able to be taken up and sucessfully transformed. For self-protecting, the frequency of heterogenous DNA transformation (butterfly wave) should be considerably low but the accumulation impact is high enough to change the whole bacterial world regarding information of genome comparing among species (tsunami).
The significance of the low frequency of heterogenous DNA transformation should not be negligible in our study since this might be the main channel through which different species communicate with one other. This communication would cause a long inevaluable impetus to the whole ecosystem in the background of the whole life history.
From the evolution perspective, homologous tranformation makes significant contribution to a special species, but make little impact to the whole ecosystem. The genetic communication between the same species is common to all over the eukaryotic and prokaryotic kindom. So, it is not surprise to discover natural transformation among bacteria of the same species with their own chromosomal DNA. However, this can not explain high frequency event of HGT among different bacterial species providing the information from comparing known genomes. Neitheir conjugation can explain this because gene communication in this form occurs only in conjugative plasmid, another special form of DNA with non-comprehensive impact. Butterfly event might edify us to find the right way to this important issue. The first step of explaning high HGT among different species is to prove that heterogenous DNA is able to be taken up and sucessfully transformed. For self-protecting, the frequency of heterogenous DNA transformation (butterfly wave) should be considerably low but the accumulation impact is high enough to change the whole bacterial world regarding information of genome comparing among species (tsunami).
The significance of the low frequency of heterogenous DNA transformation should not be negligible in our study since this might be the main channel through which different species communicate with one other. This communication would cause a long inevaluable impetus to the whole ecosystem in the background of the whole life history.
2007年5月23日星期三
Pense plus et fait moin
To assay hofQ mutant from another lab, I used hofQ OUT and hofQ IN primers. Using hofQ OUT primers, no obvious difference can be found in hofQ mutant and WT because PCR product from the mutant is less than 200 bp smaller than that from the wild type. This is comprehensible. However, the appearance of stripe which is the same as that of wild type when using primer IN demonstrated that hofQ still exist in the genome. Strangely, I also PCR amplified out the corresponding stripe which indicating the existance of cat gene. This might imply that there are not only one hofQ gene in the genome and the lab who donates me the mutant might not completely eliminate all hofQ genes in genome. Nevertheless, I will reconstruct hofQ mutant after I finish my experiments in hand.
P1 transduction est OK. Il toujour comme ca. Quand le theorie est tres complex, la experimentation est tres simple. Au contraire, quand le experimentation est tres complex, on dois réfléchir. Parce que la nature aime bien simple en action. J'ai une l'autre idée qui peut simplier la experimentation mais peut me montre la chose tres importante. Je la pense ce soir. Si il marche bien, je vais ravir. Il faut qu'on recherche comme la nature. On pense plus et fait moin.
P1 transduction est OK. Il toujour comme ca. Quand le theorie est tres complex, la experimentation est tres simple. Au contraire, quand le experimentation est tres complex, on dois réfléchir. Parce que la nature aime bien simple en action. J'ai une l'autre idée qui peut simplier la experimentation mais peut me montre la chose tres importante. Je la pense ce soir. Si il marche bien, je vais ravir. Il faut qu'on recherche comme la nature. On pense plus et fait moin.
2007年5月13日星期日
Today's expermient --Bad Luck!
I used ampicillin which was not prepared for a long time. However, lawn grow on selective plates. This must be resulted from the ineffectivity of ampicillin. I have experienced similar cases several times. I have to reprepare ampicillin tomorrow and try my experiments again.
My recent result is exiting. Because it suggested me that the rusty transformation machine works in a bacterium with different background. Does this means that the two transformation machines compete for translocation DNA IN different bacterial backgroundS?? If so, how do they compete? Which kind of mechanism controls the two? Before I know this, I have to characterize each of them. This a long long aim. Firstly, I have to recheck all my previous data firstly. The blamed ampicillin!
My recent result is exiting. Because it suggested me that the rusty transformation machine works in a bacterium with different background. Does this means that the two transformation machines compete for translocation DNA IN different bacterial backgroundS?? If so, how do they compete? Which kind of mechanism controls the two? Before I know this, I have to characterize each of them. This a long long aim. Firstly, I have to recheck all my previous data firstly. The blamed ampicillin!
2007年5月7日星期一
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