1.1. Theory – how to select?
The E. trojan is a synthetic E.coli strain that is engineered to lack an essential gene, nadE, in its genomic DNA. To survive, this strain has to rely on a pre- introduced plasmid (pDummy) bearing the essential gene; thus forcing the bacteria to maintain the plasmid until an alternative source of nadE gene is present. The pDummy, however, has been designed to have a temperature- sensitive origin of replication which would cease to function if the bacterial cells are incubated under higher incubation temperatures (>42ᵒC???).
For sub-cloning purposes, an E. trojan – compatible vector plasmid is designed. This carrier vector, like the pDummy, contains the nadE essential gene. Once a gene of interest is inserted into this vector, the plasmid can be transformed to the E. trojan for amplification. Incubating the transformed bacteria at a temperature high enough to inactivate the heat sensitive replication origin of the pDummy would result in pDummy loss, making it necessary for the cells to retain the insert- bearing pCarrier for survival. Bacterial cells that do not take up the pCarrier and its insert would be deprived of the nadE gene product and die; while those who do would survive and continue dividing.
1.2. Method of assembly
To study the population dynamics and behavior of a certain antibiotics sensitive strain of E Coli in a medium of antibiotic, our E. Trojan that is introduced into the culture medium must not process a wide spectrum of antibiotic resistance that impose a selective advantage. At the same time, E. Trojan needs to be transformed with the T4MO gene to carry out its job of signal disruption.
Summarizing the above criteria, a solution where the bacteria can be transform with the gene of interest while remaining sensitive to antibiotics is needed. Therefore the requisite is to construct a new bacteria strain that can perform plasmid selection without the use of antibiotics, and contains as little antibiotics resistance gene as possible.
Construction and maintenance of an antibiotic-resistance-gene-free plasmid through antibiotic selection – the unavoidable evil two plasmid system
Our ultimate goal is to construct our EX without conferring it any new antibiotic resistance. For this reason no resistance gene should be found in our dummy plasmid pDummy.
Yet, such a plasmid would not be maintained by itself unless the host bacterium develops an addiction to it (i.e. losses the essential gene in its genome and depends on extragenomic copies on pDummy), and inconveniently, the addiction can only be achieved after the introduction of the plasmid.
The solution is to develop a mutualistic relation between two plasmids and we planned to exploit positively regulated origin of replications. Well studied examples are those in pSC101 and R6K origins of replication, where the origins of replication (OR) appear together with a constitutive gene (G). Initiation of replication happens if and only if the trans element of the gene is provided.
Let’s consider the following scenario:
i. G is placed on pDummy with no selection marker but with a normal replication origin
ii. OR is the sole origin of replication of another plasmid (pToolkit) with a selection marker
iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress
We would obtain three possible outcomes:
1. only pDummy is uptaken
- since pDummy has no selection marker, the host bacteria die under selection pressure and cannot propagate
2. only pToolkit is uptaken
- the host bacterium that uptakes pToolkit survives. Yet during propagation, pToolkit is not replicated because proteins of G are absent. Therefore daughter cells of the host bacterium will not receive copies of the pToolkit and die under selection pressure.
3. both pDummy and pToolkit are uptaken
- in presence of pDummy, pToolkit is maintained and confers the host bacterium with stress resistance. Daughters that receive copies of both plasmids will survive and eventually develop into a colony.
Using this mutualistic relation, the desired pDummy can be maintained once the host bacterium develops an addiction it, and pToolkit can be lost in bacteria propagation if the expression of G can be shut off manually. Eventually, the bacteria not obtain any new antibiotic resistance genes but keep pDummy.
Development of addiction – use of the lambda RED recombination system
To develop the addiction in the host bacterium to pDummy, an essential gene for survival is to be deleted from the bacteria genome, provided that the bacteria can survive on extra-genomic copies after the deletion.
The deletion here is mediated through the lambda RED recombination system.
The lambda RED recombination cassette is located on the pToolkit (and hence the name of the plasmid). Once the recombination is successful, it can be eliminated from the host bacterium together with the antibiotic resistance gene.
Therefore, once the co-transformation of pDummy and pToolkit is successful, linear dsDNAs having a reporter gene flanked by homologous sequences to the essential gene can be introduced into the bacteria. When the recombination is kicked started, the essential gene will be swapped out and the reporter gene will be incorporated into the bacteria genome.
Since the linear dsDNAs do not have origin of replications, they are not inherited in daughters unless they are swapped into the genomes. Thus, any observable signals from the reporter would allow identification of successful recombination. Identified colonies can then be further treated to induce loss of pToolkit, which afterwards would be the completed strain of EX.
Complementation between reporter genes – manifesting completion of EX engineering
To ensure that the final strain of EX has: 1. successfully had its essential gene deleted from genome, 2. maintained the pDummy, a complementation reporter system between the pDummy and swapped gene is preferred over a single reporter at the swapped site.
Different methods can achieve the above aim:
i. Alpha complementation can be used in E. Coli strains where the lacZ gene is completely removed. The larger fragment ω can be swapped for the essential gene while the smaller α fragment can stay on pDummy. In a X-gal rich medium, blue colonies suggest the desired engineered strains.
ii. Complementation between split fluorescent proteins (sFP). 2010 iGEM Slovenia team has demonstrated the principle that N-terminal and C-terminal fragments of sFPS are able to complement in vivo and two sets of sfFPS are able to undergo Förster resonance energy transfer (FRET). This idea is adopted but an alternative set of candidate, split superfolder GFPs (sfGFP), was developed.
Summary of construction flow:
1. Assembly pDummy and pToolkit
2. Co-transform both plasmid into E Coli and maintain stable strains
3. Introduce linear dsDNAs and induce recombination
4. Isolate recombinants
5. Induce loss of pToolkit
1.3. Component details
Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000)
oriR101 & repA101-ts is a set of low copy origin of replication derived from the pSC101 origin of replication. The repA101-ts gene codes for a heat-labile protein that is required in trans for the initiation of replication at oriR101. In our construct, our characterization has shown that plasmids with this origin of replication can only be maintained below than 300C, and partial maintenance of plasmid was observed within temperature range from 290C to 330C. This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by a nucleotide mutation.
split superfolder green fluroscent protein_split sfGFPsplit superfolder green fluroscent
protein_split sfGFP
sfGFP1-10 (BBa_K524001)
sfGFP11 ((BBa_K524002)
The sfGFPs are mutated variants of GFPs that has improved folding kinetics and resistance to chemical denaturants. Split sfGFPs at amino acid residues 214 and 215 have been reported to undergo spontaneous complementation to give green fluorescence. The two split constructs were produced from an existing biobrick – pBAD driven sfGFP BBa_I746908. CDS of sfGFP amino acid residues 1-214 were copied out for sfGFP1-10 using PCR and stop codon was added to the end. The sfGFP11 was produced in a similar fashion, with a start codon added to the front of the CDS of amino acid residues 215 to 238.
Essential gene nadE (BBa_K524003)
nadE is a vital gene in E. Coli. It codes for NAD+ synthetase. In principle, removal of such gene from the genome would cause addiction of bacteria to a plasmid that has a copy of the gene. CyaR (a sRNA) regulates the expression of nadE post-transcriptionally. This feature is retained in our construct. Transcription of nadE operon requires the sigma-70 factor and is terminated by downstream extragenic sites. The nadE gene was cloned out from the genome of strain BL21(DE3), and was completed the nadE by having B0015 terminator assembled to its end.
Replication initiator pi protein and ori-gamma from R6K plasmid
ori-gamma is one of three replication origins (the other two being alpha and beta) of the R6K origin. Initiation of replication at ori-gamma requires the pi protein in trans, which is encoded by the pir gene. Yet doubling the concentration of pi protein would effectively shut down the replication as well. Expression of pi protein is autogenously regulated. The pir construct was cloned out from the genome of strain BW25141 (courtesy of The Coli Genetic Stock Center) and standardized. The ori-gamma was adopted from the R6K origin of replication BBa_J61001.
iGEM 2010 Slovenia Split/FRET constructs
The split CFP and YFP from the biobricks of Slovenia team last year were used as alternative reporters. The idea is to put one of the terminal fragments of a split fluorescence protein into the pDummy, and swap out the essential nadE gene from the genome with the other terminal fragment. Driven by pLac R0010, both fragments should express simultaneously when induced by IPTG and fluorescence signal would be observed as an indicator of successful recombination.
2. MIC
2.1. Theory
0. Introduction
In order to quantitatively demonstrate the effect of indole charity as well as our construct’s ability to negate it, we have decided to perform a series of minimum inhibition concentration (MIC) tests, where we subjected different strains and mixes of E.coli to an antibiotic gradient and cultured overnight (18 hours). The OD600 readings of each test were recorded afterwards and will be shown in later sections.
I. Wild Type (RR1) MIC Test
Phase 1 - Kanamycin MIC test
Experimental Design and Aim:
RR1 is a derivative from the common strain K12 and is not known to have any antibiotic resistance other than for streptomycin. Hence it was arbitrarily chosen as the non-resistant ‘wild type’ for our tests. A simple MIC test was conducted for RR1 to serve as a benchmark for comparison with later experiments, and kanamycin was opted as the antibiotic of choice. This was primarily for two reasons:
First, the kanamycin resistance gene incorporated into our selection plasmids functions through producing a mutated ribosomal protein that is insensitive to kanamycin. Unlike some other forms of resistance where antibiotic molecules are directly inactivated, this method ensures that the antibiotic levels remain relatively constant throughout the experiment, as well as prevents the appearance of satellite colonies during plating.
The other reason for choosing kanamycin is because, being an aminoglycoside, it acts by inhibiting protein synthesis through binding irreversibly to the 30S ribosome. This causes it to be bacteriostatic at low concentrations while bactericidal at high ones.
Results:
However, since we are using another untested K12 strain, namely RR1, and our growth condition is normal LB liquid culture, RR-1 MIC was carried out in the first place to confirm the MIC value for the RR-1 source we obtained.
The result of the MIC turned out to be around 7~9µg/ml, which is slightly smaller than the result indicated by the paper. The difference in their genotype could be the dominant reason while the less nutritive culture we used may affect the testing result as well.(right or wrong????need to be proved)
Phase 2 - Kanamycin MIC test with indole supplement
Experimental Design and Aim:
Indole has been proposed as a key signalling molecule produced by unstressed (high resistant) E. coli as a form of ‘charity’ that grants stressed (low resistance) cells passive immunity against antibiotics. This enables such stressed individuals to continue to survive and proliferate. Indole functions by inducing the expression and activity of multidrug efflux pumps to expel antibiotics and toxins, as well as activating oxidative-stress protective mechanisms to minimize DNA damage.[1] In an attempt to prove and quantify this effect, we repeated the kanamycin MIC test, this time supplementing the LB medium with different concentrations of indole, ranging from 300µM to 2mM.
Results:
The effect of indole on the MIC for RR-1 various under different concentration. Naturally, the indole production of E.coli is around 300µM while under antibiotic stress, the production will decrease to undetectable level.[1] We think the indole concentration of low and high resistance mixed culture should be around 300µM as well. However, the optimal concentration for charity work is still unknown.
For the testing under indole concentration of 300µM and 500µM, we can see that the MIC for RR-1 increased to ( )which is in consistence with the result of our Mixed culture MIC posted later. The RR-1 is able to survive under kanamycin concentration of ( ) .
On the other hand, we also did some 1mM and 2mM indole MIC testing, which aims at finding out whether the over dosage of indole could kill the population instead of protecting them. The result shows that indole did have a killing effect at higher concentration and the MIC did decrease compared to the result of 300µM indole MIC.
II. Mixed Culture MIC Tests
Phase 1 - Wild type (RR1) with RFP-labelled kanamycin resistance strain (RFP) (99:1)
Experimental Design and Aim:
As metioned previously, when E. coli cultures are subjected to antibiotic selection pressure, a small number of naturally resistant individuals, at some cost to themselves, provide protection to other more vulnerable cells by producing indole, resulting in an overall enhancement of the survival capacity of the population in stressful environments. To mimic this naturally occurred phenomenon, a kanamycin resistant strain, which represents the mutants, was introduced into the RR-1 at 1:99 ratio. This kanamycin resistant strain was labeled with RFP for easy recognition. The ratio of kanamycin resistant strain, KanR/RFP, to RR-1 was recorded for later comparison with that of later mix culture assays.
Results:
We can clearly see the effect of the charity work from our result. Even under kanamycin concentration of 25µg/ml, which is half of the working concentration of kanamycin and almost 3 folds of RR-1 MIC, RR-1 is still growing rapidly and maintain the majority of the overnight culture. The OD600 result didn’t show a clear co-relation with kanamycin concentration and is floating around 1.1.
The column chart also shows that the ratio of RFP to RR-1 falls between ½ and ⅓ after overnight culture. This ratio may change when the kanamycin concentration approach its working concentration. We plan to prove this in our future testing.
Phase 2 - Wild type (RR1) with kanamycin resistance T4MO (GRP)
Experimental Design and Aim:
In order to interfere the indole charity work and obtain a more efficient selection, we introduce a plasmid which encodes Toluene-4-Monooxygenase (T4MO), an enzyme that catalyzes the oxidation of indole into indigo. To test its effect, we design a T4MO/KanR and RR-1 (1:1) mixture culture MIC test. In this test, we assume that the indole degradation rate of T4MO will be close to the indole producing rate of itself along with the mutated minority in RR-1, which means a lower MIC of RR-1 will be observed in comparison of the KanR/RFP and RR-1 mixculture due to the absence of the indole charity work. The ratio of T4MO/KanR to RR-1 was also kept in the testing for later comparison with that of the 3 way mix culture assay.
Results:
Since the charity work is simply being weakened by the introduced enzyme, RR-1 is still growing quite well under kanamycin concentration of 25µg/ml. However, the ratio of the two strain, T4MO/KanR and RR-1, is different from former result from mixed culture of RFP/KanR and RR-1. Under lower kanamycin concentration, RR-1 still remain to be the majority of the culture and the difference is not very obvious. When kanamycin concentration exceeds 10µg/ml, we can see that T4MO out-competed RR-1 and became the majority of the overnight culture. Comparing with the ratio got from RFP&RR-1 mixed culture, we can draw the conclusion that the charity work of is weakened and the efficiency of ( )
Phase 3 - Wild type (RR1), RFP-labelled kanR strain and GFP-Labeled KanR T4MO (98:1:1) [???]
It has been proved in the phase 2 that T4MO does interrupt the indole charity work. So in the next step, we plan to practice our model, which is introducing a T4MO strain into the environment predominantly consisting of RR-1with few kanamycin resistant mutants. By comparing the resulting ratio of RR-1 to the antibiotic resistant strain to that of the T4MO and RR-1 Mix culture, we may observe again the strong effect of indole; by comparing the resulting ratio of RR-1 to the antibiotic resistant strain to that of the KanR/RFP RR-1 Mix culture without T4MO, we would be able to tell how T4MO takes effect.
III. Conclusion
[lalaala]
IV. Future Plans
Phase II - Wild type (RR1), RFP-labeled kanR, and GFP-labeled T4MO/Bcr mix
Our ultimate goal is to boost the selection efficiency by introducing a T4MO/Bcr strain, which can interfere with the indole charity work. The Bcr gene, which encodes a multidrug pump, keep this strain survive and produce T4MO. By adjusting IPTG concentration, this strain will keep working for a certain period of time and die afterwards as to the accumulation of kanamycin inside the cell. However, as we didn’t have time to characterize the efficiency of Bcr, and another essential part of our project, the alternative selection method, is still in progress yet, we are unable to do this construction and perform further testing.
(insert a picture here showing out ideal construction)
By having this strain in the population, the charity work will be restricted, so that the selection process can be done efficiently without applying over dosage of antibiotics, and the presence of this strain can be controlled by us so that this alien strain only performs the duty of degrading indole and brings no side effect on the whole selection process.
V. Biobrick construction
Bcr
Bcr is a type of multidrug efflux pump, which are integral membrane proteins that utilize cellular energy to extrude antibiotics or biocides actively out of the cell. It belongs to the major facilitator superfamily (MFS), and is known to contribute to multidrug resistance in E. coli.
Under normal growth conditions, a large number of drug efflux pumps are thought to be weakly expressed. In particular, literature documents Bcr to confer varying degrees of resistance to several kinds of antibiotics when overexpressed; including bicyclomycin (selection-capable), tetracycline (8-fold MIC increase*), and kanamycin (4-fold MIC increase*).
In our iGEM project, we planned to construct a biobrick with the pLac promoter driving expression of Bcr. The reason behind this is to take advantage of the additive effect of IPTG on pLac activation. We hope that by varying the concentration of IPTG, we can control the level of expression of Bcr and thus manipulate the mutant E. coli’s MIC to certain antibiotics.
However, as the time limited,we only submit a plasmid contain only the bcr gene to part registry. You can use it for selection of bacteria in the future.
*: compared with wild type
VI. Appendix
[Extra data] Protocols will probably be included in the Notebook section
[1] (http://www.nature.com/nature/journal/v467/n7311/abs/nature09354.html)
[2](http://www.scielo.br/pdf/gmb/v26n2/a17v26n2.pdf)
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