Team:CAU China/Results
Results
Part I: Make Corynebacterium glutamicum produce polyglutamic acid, and measure the expression of P0864 promoter.
Work we have finished
1.Determination of constitutive promoter (P0864(BBa_K3796000))strength
P0864(BBa_K3796000) is a constitutive promoter in Corynebacterium glutamicum which had little information previously on the Registry. We use this promoter to control the expression of the γ-PGA synthetase genes in our project. Therefore, we need to test its strength in C. glutamicum first. Also, to fully characterize this new part, we want to know whether it works in the typical chassis E. coli as well.
We want to achieve this goal by inserting a reporter gene after P0864 to visually show its strength as a promoter. For convenience, we choose the fluorescent protein mCherry, whose excitation light and emission light both fall into the range of visible light. Also, we choose to use the shuttle vector pXMJ19 that is applicable to both E. coli and C. glutamicum. The genetic circuit we use as a device in testing is BBa_K3796219 and the blank control is BBa_K3796220. Particularly, you may notice that there is a tac promoter upstream of the MCS on pXMJ19, so a proper blank control to exclude the influence of Ptac leaky expression is needed. Ideally, the fluorescence intensity/OD600 should be higher in the experimental group than in the control group.
Figure 1 Genetic circuit for testing the strength of P0864
Figure 2 Genetic circuit for blank control
We built the genetic circuits using Clonexpress® Multi One Step Cloning kit from Vazyme to connect RBS sequence (BBa_K3796001, synthetic) and mCherry gene (BBa_J06504) downstream to P0864. We successfully constructed the above sequences and verified them by colony PCR and sequencing.
Figure 3 Agarose gel map of BBa_K3796219 circuit.
a.Lane 1 to lane 9 are electrophoresis bands of BBa_K3796219 circuit, where lane 1 to lane 4 refer to transformed E. coli, lane 5 to lane 9 refer to transformed C. glutamicum. b. Lane 1 (E. coli) and lane 2 (C. glutamicum) are electrophoresis bands of BBa_K3796220 circuit
In the 1st iteration, we tested the strength of P0864 by a simple qualitative test of the fluorescence under the excitation light of mCherry protein (580nm).
Figure 4 The difference of fluorescence in Escherichia coli (including experimental group (left) and control group (right)).
a. Image under excitation light. b. Image under natural light.
Figure 5 Difference of fluorescent proteins expressed by Escherichia coli and Corynebacterium glutamicum.
a. Images of Corynebacterium glutamicum after centrifugation, including experimental group (left) and control group (right). b. Images of E. coli after centrifugation, including experimental group (left) and control group (right).
As you can see, compared with the control group, the experimental group showed red fluorescence apparently, both under the natural light and excitation light. However, qualitative tests are not enough for the characterization of the strength of P0864, and we further wanted to know in which chassis did it work better.
We designed a qualitative test to better characterize this promoter in different chassis. After the bacteria had been cultured at 37 ℃ for 26 hours, the fluorescence was measured with a fluorescence spectrophotometer and OD600 was measured with a visible spectrophotometer. We use the ratio of fluorescence intensity/OD600 to reflect the relative fluorescence intensity under the unit absorbance value.
Figure 6 After E. coli and C. glutamicum were cultured at 37 ℃ for 26 hours, the ratio of measured fluorescence intensity data to OD600 data.
(the excitation light of 580nm and emission light of 610nm)
T-tests were performed to show that there was a significant difference between the experimental group and the control group. Plus, this difference is bigger in C. glutamicum. We have done four repeats and the standard deviation is acceptable.
We learned P0864 is a constitutive promoter that works both in E. coli and C. glutamicum. Its strength tends to be stronger in C. glutamicum. If used in E. coli, codon-optimization may be carried out.
2. Modular construction of capA capB and capC
For each gene in the capABC gene cluster, we connected P0864, RBS and terminator sequences upstream and downstream of them.
Figure 7 Modules need to be constructed.
a. capA module;
b.capB module;
c.capC module
Figure 8 Agarose gel electrophoresis of capA capB capC modules.
a. capA module.
b.capB and capC modules. Lane 1 to 6, 8, 9, 10, 12: capB module, lane 11, 13: capC module
Work we have not done yet
Because the length of the module is large, and the head and tail sequences of the three fragments are the same, we designed different methods to add specific marker sequences to the head and tail, and tried to integrate the capA module, capB module and capC module by seamless cloning or enzyme digestion, but all failed.
Figure 9 The agarose gel electrophoresis of PCR(capC)
We amplified the capC module to test the connection of the three modules.
We tested the transformed bacteria (E. coli DH5α) and the colony PCR verification was carried out. The target band shown in the figure was the capC module. In the detection, 12 plaque bands could be amplified, of which 6 bands were obviously bright. However, we could not extract the corresponding plasmids from these bacteria.
Conclusion
At present, we have completed the determination of P0864 expression in E. coli. However, it was not successfully transformed into Corynebacterium glutamicum.
We completed the construction of each gene module of capABC, but failed to connect the three modules.
Failures & Difficulties
In the experiment, we encountered many difficulties in construction and detection, as well as the difficulties from COVID-19. Further, our experiment was short of funds. In this case, many kits could not be purchased in time. Therefore, we summarized a relatively simple identification process after recombinant plasmid transformation: colony PCR → Plasmid Extraction → Sanger sequencing (test pass or one-way sequencing). We omitted the step of enzyme digestion verification, which consumed a large amount of enzyme and could not avoid the step of Sanger sequencing. The companies that cooperate with us in sequencing include Sangon and Tsingke. In addition, in the case of lack of funds, we also reduced the amount of nucleic acid dye as much as possible. Each time we used agarose gel, we used 20ml 1× TAE buffer, add 2 μL nucleic acid dye. Nucleic acid dyes still have good dyeing ability at this dosage, and can significantly reduce the dosage.
Part II: Transformation of Corynebacterium glutamicum metabolic pathway. The expression of ppc and odhA was enhanced and decreased α-Ketoglutarate dehydrogenase expression
Work finished
1.odhA
In order to improve the production of glutamic acid which is formed from the citric acid cycle intermediate α-ketoglutarate, we decided to attenuate odhA which encodes KGDH that catalyzes α-KG to form succinyl-CoA formation to introduce more carbon fluxes to glutamic acid synthesis rather than to the succinyl-CoA formation. So we designed the experiments of introducing a weaker strength RBS to the upstream of its ORF for odhA attenuation.
Figure 10 Sequence for modifying RBS upstream of ppc in C. glutamicum genome
Construction of Recombinant Plasmid pK18mobsacB-RBS-odhA
The first thing to introduce a weaker strength RBS to the upstream of its ORF is constructing the recombinant plasmid pK18mobsacB-RBS-odhA successfully. The weak RBS addition step was completed in the odhA amplification stage. To achieve this goal, we decided to amplify odhA by dividing it into odhA-UP and odhA-DOWN to amplify.
Figure 11 The agarose gel electrophoresis of PCR (odhA-UP) Lane MK: Marker; Lane1: the target band of odhA-UP
Figure 12 The agarose gel electrophoresis of PCR(odhA-DOWN) Lane MK: Marker; Lane1: the target band of odhA- DOWN
After amplification of odhA-UP and odhA-DOWN, we digested pK18mobsacB by SmaI, and fused odhA-UP, odhA-DOWN and SmaI-pK18mobsacB to construct the combinate plasmid by One Step Cloning Kit---ClonExpressTMMultiS. Then the combination plasmid was transferred into DH5α for plasmid amplification.
In order to identify whether the combination plasmid was constructed successfully or not, we used three ways to verify it. The first method was colony PCR whose template was the exposed DNA after cell pyrolysis (Figure 4). Secondly, through enzyme digestion verification we observed the target (Figure 5). Finally, we extracted the plasmids in DH5α, sequenced them and the response was correct.
Figure 13 The agarose gel electrophoresis of colony PCR to verify recombinant plasmid(pK18mobsacB-RBS-odhA)
Figure 14 The agarose gel electrophoresis of the enzyme-digested product(SmaI)
Lane MK: Marker; Lane1: the band of pK18mobsacB-RBS-odhA; Lane1: the target band of pK18mobsacB-RBS-odhA disgested with SmaI
Transformation of Corynebacterium glutamicum ATCC 13032
The following step after successfully constructing the pK18mobsacB-RBS-odhA combination plasmid was to transform it into Corynebacterium glutamicum ATCC 13032 by electroporation (Figure 6). Because pK18mobsacB is a mobilizable vector with KmR and sacB allowing for selection of double crossover in C. glutamicum, we default C. glutamicum has occurred the first exchange if it can grow on the LBG-Kan solid medium. In order to further verify the first exchange has already happened, we performed colony PCR validation, and the result displayed that the 9th strain has the target fragment.
Figure 15 Transformation of Corynebacterium glutamicum ATCC 13032
Figure 16 The agarose gel electrophoresis of colony PCR to verify the first exchange.
Lane MK: Marker; Lane1: positive control; Lane2: the target band
2.ppc
In order to construct a strain with high glutamic acid yielding, we have decided to achieve this goal through the double-crossover replacement technique with plasmid pK18mobsacB, a plasmid containing both kanamycin resistance and sacB for double screening. PEPC encoded by ppc in the TCA cycle catalyzes PEP to form OAA. By overexpressing ppc through an RBS replacement, we expect a growth in glutamic acid production in our recombinant strain.
We have constructed a recombinant plasmid pK18mobsacB-RBS-ppc for the RBS replacement in Corynebacterium glutamicum ATCC 13032, the results are as follows.
Figure 17 Amplification of ppc-UP.
Lane: MK: Maker; Lane1: ppc-UP.
Figure 18 Amplification of ppc-DOWN.
MK: Maker; Lane1: ppc-DOWN.
Figure 19 Restriction digestion of pK18mobsacB with SmaI.
MK: Maker; Lane1: SmaI-pK18mobsacB.
Figure 20 Colony PCR.
Lane: MK: Maker; Lane1: positive control; Lane2: colony PCR product.
Figure 21 Double digestion with XbaI and EcoRV.
MK: Maker; Lane1: pK18mobsacB-RBS-ppc; Lane2: double-digested product.
What’s more, we further delivered the plasmid from positive strains selected above to sequencing and were able to confirm the correct target plasmid.
Work unfinished
1.odhA
Verification of the second exchange of double crossing
After verifying the first exchange has occurred, the 9th strain was cultured in the LBG solid medium to induce the second exchange, but when we verified by using resistance screening, it failed.
Phenotypic verification
We plan to do the activity test of enzyme ODHA and the glutamic acid production test to verify our experiment-odhA attenuation.
2.ppc
With the successful construction of recombinant plasmid pK18mobsacB-RBS-ppc, we then attempted to transform it into the competent cells of Corynebacterium glutamicum ATCC 13032. However, we have not screened any positively recombinant strains yet. For the future, if we could successfully construct a recombinant strain with the RBS change in Corynebacterium glutamicum ATCC 13032, we would be able to carry out measurement of enzyme activity and glutamic acid production assay.
3.RNAi
We plan to reduce enzyme activity by RNAi technology, prevent the degradation of γPGA by bacteria, and finally achieve the goal of producing γPGA.
Figure 22 RNAi circuit
Construction method:
By designing a suitable primer, the anti enzyme DNA sequence with RBS is amplified from the Corynebacterium glutamicum. The sequence is connected to the plasmid by Restriction enzyme digestion, and the recombinant plasmid is imported into E. coli to verify the success of plasmid construction. If successfully constructed, the recombinant plasmids will be imported into Corynebacterium glutamicum to complete the transformation of engineering bacteria.
Conclusion
1.odhA
a) We first completed the experimental design of this program. Then, through the technical means of molecular biology, we completed the construction of the recombinant plasmid pK18mobsacB-RBS-odhA. After that, we carried out the transformation of Corynebacterium glutamicum ATCC 13032.
b) In order to truly complete the transformation of glutamic acid to increase production, in the future, we will perform phenotypic verification by the activity test of enzyme ODHA and perform the glutamic acid production test to ensure that the plasmid we introduced really plays a role in Corynebacterium glutamicum.
2.ppc
With the successful construction of recombinant plasmid pK18mobsacB-RBS-ppc, we then attempted to transform it into the competent cells of Corynebacterium glutamicum ATCC 13032. However, we have not screened any positively recombinant strains yet. For the future, if we could successfully construct a recombinant strain with the RBS change in Corynebacterium glutamicum ATCC 13032, we would be able to carry out the measurement of enzyme activity and glutamic acid production assay.
3.RNAi
The first round of construction is currently under way after the introduction of E. coli electrophoresis verification failed, and the second round of rebuilding is under way. The second round of builds is expected to be completed by the deadline.
In the future, we need to be determined to build successfully on the basis of the anti sequence into Corynebacterium glutamicum, and the gene is integrated into the genome so that the interference effect can be stable in a long term.
Failure & difficulties
1.odhA
- The effect of PCR under theoretical conditions is not up to expectations. Through gradient PCR, we have improved the PCR conditions for extracting target fragments to ensure that sufficient target fragments are obtained.
- Seamless cloning and electrotransfer require multiple attempts before success.
- The selection of homologous recombination steps has been adjusted many times. For example, the initial scheme was positive screening and negative screening. After several experiments failed, after discussion, we adjusted the screening method to relaxation culture.
2.ppc
At first, our amplification of ppc-UP and ppc-DOWN failed. Considering the high GC content of our target strain, we changed the primers and conducted gradient PCR to determine the proper annealing temperatures, and finally managed to overcome the setbacks.
The transformation of Corynebacterium glutamicum ATCC 13032 failed. Furthermore, another successfully constructed recombinant plasmid using the backbone of pK18mobsacB was not able to transform Corynebacterium glutamicum ATCC 13032 either, while constructions using the backbone of pXMJ19 succeeded. We are not sure that if the problem is caused by the backbone plasmid yet, but we hope to conduct more experiments by changing the backbone for another round of construction and so on in the future.
3.RNAi
The RBS area was not designed during the first design sequence, resulting in ineffective previous experimental work. It remind us to think hard before building a target fragment, and make sure the experiment design is correct before experimenting.
Part III: Kill switch construction
Work Finished:
1.Verified that PgsiB can respond to salinity changes in C. glutamicum
We aim to verify that the promoter PgsiB, originally from Bacillus subtilis, can respond to salt stress in Corynebacterium glutamicum and we also want to document its response towards different salt concentrations.
Figure 23 Gene circuit for PgsiB verification
To characterize its response, we built a plasmid pXMJ19-PgsiB-gfp using the ClonExpress II one-step cloning kit (Vazyme Biotech, China) in order to test the function of PgsiB by the fluorescence of GFP. Particularly, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence.
Figure 24 The agarose gel electrophoresis of PCR product of pXMJ19-PgsiB-gfp transformed E. coli
Lane: MK: Marker; Lane1: colony PCR product. Target: PgsiB-gfp (818bp)
Figure 25 The agarose gel electrophoresis of PCR product of pXMJ19-PgsiB-gfp transformed Corynebacterium glutamicum
Lane: MK: Marker; Lane1:colony PCR product. Target: PgsiB-gfp(818bp)
After sequencing and amplifying, that vector, as well as unmodified pXMJ19, were transferred into Corynebacterium glutamicum separately and cultivated on plates. After cultivating them in the LB liquid culture medium at 100 rpm, 30℃ for 12h, the OD600 of the two bacteria were adjusted to nearly the same(nearly 2.0) and inoculated in the salt gradient LB liquid medium. After cultivation of 24h, the bacterial solution was collected and washed with PBS. Then we measured its fluorescence intensity by HITACHI F-7000, a fluorescence spectrophotometer, according to the excitation light of 488nm and emission light of 507nm and OD600. Data are shown below:
Figure 26 Fluorescence intensity results in the verification of PgsiB (the excitation light of 488nm and emission light of 507nm)
We use the control group (bacteria with empty vector) to exclude the influence of the fluorescence bacteria originally have. Through t-test, we know there is a significant difference between the control group and the experiment group, proving our results valid. Data shows that the difference of relative fluorescence intensity(fluorescence intensity/OD600) between the two becomes bigger as the salt concentration increase, indicating that there is a higher expression of gfp under the control of PgsiB when the salt concentration is high.
We finally come to the conclusion that PgsiB can respond to salinity changes in C. glutamicum, and high salt concentration can improve the expression of the downstream genes of PgsiB. Three parallel experiments were done later, which supported our conclusion. However, we haven’t observed a typical turning point in our curves, and we want to do more experiments about that in near future.
2.Verified that Patp2 can respond to pH stress in C. glutamicum
We aim to verify that the promoter Patp2 can respond to pH stress in Corynebacterium glutamicum and we also want to document its response towards different alkalinity.
Figure 27 Gene circuit for Patp2 verification
To characterize its response, we built a plasmid pXMJ19-Patp2-gfp using the ClonExpress II one-step cloning kit (Vazyme Biotech, China) in order to test the function of Patp2 by the fluorescence of GFP. Similar to verification of PgsiB, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence.
Figure 28 The agarose gel electrophoresis of PCR product of pXMJ19-Patp2-gfp transformed E. coil
Lane: MK: Marker; Lane1: colony PCR product. Target: Patp2-gfp (825bp)
Figure 29 The agarose gel electrophoresis of PCR product of pXMJ19-Patp2-gfp transformed Corynebacterium glutamicum
Lane: MK: Marker; Lane1: colony PCR product. Target: Patp2-gfp (825bp)
We incubated the bacteria of the control group and experiment group with the similar method we used in the verification of PgsiB, just changing the salt gradient to alkalinity gradient LB liquid medium. Then we measured its fluorescence intensity by HITACHI F-7000 according to the excitation light of 488nm and emission light of 507nm and OD600. Data are shown below:
Figure 30 Fluorescence intensity results in the verification of Patp2 (the excitation light of 488nm and emission light of 507nm)
Through t-test, we can know that there’s a significant difference between the control group and the experimental group under different pH conditions. The relative fluorescence intensity of the control group is relatively stable at the pH range of 7-9.5, while there is a significant increase of relative fluorescence intensity in experimental group. Data shows that the difference of relative fluorescence intensity between the two groups remains high between pH=8.5 and pH=9.0, and reaches its peak near pH=9.0, indicating that the promoter Patp2 has the most ability to enhance its downstream gene expression in this pH range. Also, we can see a sharp drop in the relative fluorescence intensity in the experiment group when the pH is higher than 9.5. We assume that this is because C. glutamicum can’t tolerate such high alkalinity stress and the OD600 decreases a lot, making the relative fluorescence intensity seems abnormal or irregular.
Three parallel experiments were done later, which supported our opinions. We finally come to the conclusion that Patp2 can respond to pH changes in C. glutamicum, and high alkalinity can improve the expression of the downstream genes of Patp2. According to our data, the peak occurs near pH=9.0, which happens to fall in the range of the alkalinity of saline-alkaline soil.
3.Verified that ndoA has a killing effect in C. glutamicum
We aimed to test if the over-expression of the toxin gene ndoA from Bacillus subtilis can kill Corynebacterium glutamicum effectively as well.
Figure 31 Genetic circuit for ndoA verification
To characterize its killing effect, ndoA is inserted into the plasmid pXMJ19 to construct a circuit with tac promoter, lac operator, ndoA and terminator rrnB using the ClonExpress II one-step cloning kit (Vazyme Biotech, China). The expression vector is transformed into E. coli DH5α first and then into C. glutamicum by electroporation.
Figure 32 The agarose gel electrophoresis of enzyme-digested product. (a)(Hind Ⅲ) and Colony PCR (b)(E. coli).
Lane(a): MK: Marker; 1:pXMJ19-ndoA digested by Hind Ⅲ; Lane(b): MK:Marker; 1:Product of Colony PCR (pXMJ19-ndoA in E. coli)
Figure 33 The agarose gel electrophoresis of Colony PCR (C. glutamicum).
Lane: MK:Marker; 1,2:product of colony PCR (pXMJ19-ndoA in C. glutamicum)(Targets are respectively endogenous mazF and ndoA); 3,4:negative control
To design a quick qualitative test, a ‘divided plate’ assay was carried out at the beginning. We divided a plate with LB medium into four parts and use the spread plate method to see if ndoA can kill C. glutamicum and this killing effect is not caused by the toxicity of IPTG. Bacteria carrying empty vector was added into 1 Ep tube and bacteria carrying the gene circuit was added into 2 Ep tubes with 1.8 mL LB liquid containing 10 μg/mL chloramphenicol respectively. After incubating the culture in a shaker at 30 °C, 220 rpm until OD600 reached 0.6, we prepared LB plates that were separated into four quarters marked A, B, C, D. We spread the diluted bacteria solution on the quarters respectively, and all plates were incubated at 30 °C for a certain time.
Figure 34 Divided Plate Assay
Quarter A: C. glutamicum carrying empty vector and 0.8mM IPTG was added; Quarter B: C. glutamicum carrying empty vector without adding 0.8mM IPTG; Quarter C: C. glutamicum carrying the vector pXMJ19-ndoA with 0.8mM IPTG; Quarter D: C. glutamicum carrying the vector pXMJ19-ndoA without 0.8mM IPTG.
Comparing Quarter A and Quarter B, we can see that the growth condition of the two is very similar, which means that the toxicity of 0.8mM IPTG is very low and it hardly kills C. glutamicum. Comparing Quarter C and Quarter D, we can see that there are apparently fewer bacteria survived in Quarter C, and the diameter of the colonies in Quarter C is rather small as well. As for the comparison between Quarter B and Quarter D, we assume that there is a serious leaky expression of ndoA controlled by tac promoter in Quarter D, which deteriorates its growth condition. We repeated the experiments 5 times and got nearly the same result. Hence, we can get the preliminary conclusion that ndoA does have a killing effect on C. glutamicum.
To give a further qualitative test, we carried out a CFU assay to characterize the killing effect of ndoA instead of determining OD600 in order to get rid of dead bacterial cells. CFU was quantified by counting the colonies on one plate and normalizing the number to volume of 1 mL culture. We added 0.8mM IPTG as OD600 reached 0.6, and estimate CFU by spreading 25μL of the culture on 3 LB solid medium every hour after the induction with 0.8mM IPTG, and our results are as follows.
Figure 35 Results of the CFU Assay, plotted against induction
It is visually discovered that the number of colonies carrying pXMJ19-ndoA fell off in the presence of IPTG, while the same bacteria grew well without induction within the first four hours and then its CFU decrease probably due to the leaky expression. We can also see that the growth condition of the bacteria carrying the empty vector hasn’t been affected by 0.8mM IPTG, since its CFU curve increases as normal. We repeated the experiments 3 times and all the curves show similar trends.
Therefore, we can finally come to the conclusion that the ndoA does have a killing effect on C. glutamicum, and can be used in our project as the toxin gene.
Work Unfinished:
Construction of the final composite kill switch and test its effect:
We are currently building the final composite kill switch (BBa_K3796209) since our verification experiments proved the basic parts we plan to use could work as expected. After the construction, we aim to test if this kill switch can kill bacteria effectively only when the pH and salinity of the environment both decrease to an expected level.
To take full advantage of the gene circuits we have already built, we are using restriction enzyme digestion to replace the gfp with lacIq in BBa_K3796206 and BBa_K3796207. The part BBa_K3796217 can be used directly in the final construction. Next, we will use CloneExpress® MultiS One Step Cloning Kit to build the final circuit.
Conclusion:
We have verified that the promoter PgsiB can respond to salt stress and the promoter Patp2 can respond to alkali stress. They both work in our chassis, Corynebacterium glutamicum. Also, we have verified that the toxin gene ndoA from Bacillus subtilis is toxic for the growth of Corynebacterium glutamicum as well.
In the future, we will continue to construct the final composite kill switch (BBa_K3796209) using seamless cloning and test its effect by incubating the bacteria in different salinity and pH and measuring CFU.
Failure and Difficulties:
In our kill switch experiments, we have met several difficulties:
- The multiple cloning site (MCS) in the plasmid pXMJ19 is a bit weird. There’s a short nucleotide (6 amino acids) in the first half of this MCS, meaning that any insertion of genes behind this coding sequence is likely not to be translated. We found this error in August, which meant we have to re-build our gene circuits……
- Though we are using lacIq to make Ptac strictly under control, we are still facing serious leaky expression, which makes our verification experiments about ndoA very difficult. In the future, we plan to change the inducible promoter to a stricter one, and then re-build our gene circuit to test ndoA to get better results.
- In the promoter verification experiments, it is hard to find a proper container to cultivate nearly twenty tubes of bacteria. At first, we tried 50-ml- centrifuge tube, worried about bacteria growth. However, they grow so well that we have to increase the dilution ratio to get a suitable concentration for measuring its fluorescence intensity. Besides, it is also a waste of tubes. Thus, for the second time, we tried 2mL centrifuge tubes, after an observation of bacteria growth which indicated that bacteria can also grow pretty good if they have plenty of air to breathe, 1mL in 2mL centrifuge tube, for an instant. Of course, using microplates can be a better choice, if you have a relative microscale fluorescence spectrophotometer.
- We have tried to use restriction enzymes to build our gene circuits but we met countless failures and wasted lots of time. We now think using seamless cloning is a better choice to build circuits efficiently.