Team:YiYe-China/Contribution
1. Characterization of a related previous part BBa_K3577001supplemented with new data.
2. Document troubleshooting that would be helpful to future teams.
1. New data
Objective
Part K3577001 is a part constructed by the iGEM Worldshaper-Shanghai team in 2020.This year, We’d like to give future iGEM teams more information about this part so that everyone can understand it better and may use it in future projects.
Methods
Based on K3577001, plasmid pBS1C3 contain trigger was constructed. Plasmid pBS1C3 with toehold-mcherry and pCOLAD-trigger were transformed into E. coliBL21(DE3) strain.Single colony was selected to inoculate LB broth containing kanamycin and cultured overnight.
Results
As shown in the figure 1, the expression of fluorescent protein can be observe in E.coli.And the fluorescence value of only toehold is much lower than that of toehold with trigger, which shows that only toehold can not cause fluorescence,but with trigger lead to a very different fluorescence value（Fig.2). Prove that the toehold system can work to open the hairpin cause a fluorescence.
According to our experimental results：
1. Compare with the control group, there is obvious red light, and the fluorescence value is much higher than others, which is consistent with the initial results of this part.
2. Only very low fluorescence values occur in toehold alone，due to a small amount of leakage,a few fluorescent signals is generated however, it has been shown that the fluorescence value of toehold and trigger is very different from the toehold alone, and the value is much higher than the toehold alone, which proves that its toehold system is working properly.
2.Document troubleshooting
People who have done molecular cloning experiments might know that we always encounter various problems during the experiment. However, these problems can generally be solved. We have sorted out the common problems and solutions in molecular cloning experiments in iGEM competitions based on our own experimental experience and some data. We mainly talk about the problems that would happen during the process of PCR, Restriction Enzyme Digestion, and Ligation and Transformation. We hope this troubleshooting can be a helpful resource for the future iGEM teams.
2.1 PCR
The polymerase chain reaction (PCR) is a technique that amplifies a DNA template to produce specific DNA fragments in vitro.
Unexplainable failure of amplications
●Insufficient Mg2+ ions: Insufficient or omitted magnesium will result in no or reduced PCR product. Use 1.5 mm Mg2+ ions in the final reaction.
●Poor quality of primer: Check if the primer has the correct sequence and is complementary to the template. Use primer design programs to avoid repetitive sequences, regions with high complementarity, etc. Also, BLAST search can help to avoid primers that may amplify pseudogenes or may trigger unexpected regions.
●Suboptimal annealing during thermal cycling [1]
■Optimize the annealing temperature stepwise by 1–2°C increments, using a gradient cycler when available. The optimal annealing temperature is usually 3–5°C below the lowest primer Tm.
■Adjust the annealing temperature when a PCR additive or co-solvent is used.
■Use the annealing temperature recommended for a specific DNA polymerase in its optimal buffer. Annealing temperature rules for primer sets can vary between different DNA polymerases.
Nonspecific Bands or Primer-Dimers
●The primer contains some impurities: The impurities in primers may inhibit PCR. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 μM of each primer. If so, we suggest you use some desalted primers or more highly purified primers.
●Too much Mg2+ was added: The high concentrations of magnesium increase the likelihood of nonspecific primer binding and unwanted product formation. Reducing the amount of magnesium in the final reaction.
Smeared Bands
●Too much template was added: If the template concentration is too high, the polymerase can be inhibited due to the carryover of inhibitors or inefficient denaturation. To solve this problem, you can try to reduce the number of cycles, reduce the template concentration, and/or increase denaturation time/temperature.
●The template may contain an exonuclease or was degraded. Use a fresh template.
Restriction Enzyme Digestion is a technology to cut DNA by using restriction enzymes, and cleavage can produce sticky ends (5′ or 3′ protruding ends) or blunt ends.
unexpected cleavage of the substrate DNA
●This may be due to mutations in the substrate DNA during propagation and amplification. A mutation may destroy a known restriction site or create a new one.
□Recheck cloning strategies and ligated sites. Newly generated restriction sites in DNA constructs may be overlooked.
Using Sanger sequencing of the DNA to determine whether a mutation is the cause of unexpected cleavage of the substrate DNA.
Incomplete or no digestion
●Inactive enzyme: First of all, you need to check the enzyme’s expiration date. Then, make sure you store the enzyme at –20°C. Below this temperature, the enzyme may freeze and become inactive. There are some cautions you need to be aware of:
○Use a benchtop cooler to store and transport enzymes.
○Do not store the enzyme in a frost-free freezer or the freezer door shelves.
●Restriction enzymes can easily catch fire. It is recommended to reconfirm with new unsealed enzymes.
●The efficiency of restriction enzyme digestion does not increase when there are more restriction enzymes added. You need to always follow the ratio of enzymes that are required strictly and do not change the ratio of enzymes.
Ligation is the step that joins the insert and vector once the fragments of interests are obtained.
At the end of the experiment, if you find a problem of few or no transformants, there might be some problems that happened during the ligation step.
●Poor ligation efficiency
○If you meet the problem of less efficient ligations, as with blunt ends DNA fragments, the addition of inert macromolecules like polyethylene glycol (PEG) is recommended to increase the effective concentration of reaction components and thus improving the ligation efficiency.
○Check and optimize ligation parameters like insert-vector molar ratios, reaction temperature, and reaction time.
■In order to improve the outcome of ligation, people generally set up multiple reactions with the range of 1:1 to 5:1 insert vector molar ratios. There are some apps you can find online that can help you to access convenient tools and calculators(the vector to insert molar ratios calculator).
■The range of ligation temperature is from 14°C to 25°C, and the optimum temperature is 16°C. The reaction time is typically from 10 minutes to 16 hours. In general, a higher reaction temperature requires less time but may produce a lower yield. ).
Self-ligation of vector
●If there are many transformants, but they are all wrong or original plasmids, it is very likely that the vector has not been cut cleanly. We recommend doing enzyme digestions again.
●Since the vector is prone to self-ligation after digestion, alkaline phosphatase can be used which can prevent the vector from self-ligation and improve the ligation efficiency. The specific step is to add 1ul NEB's clip into the vector and then treated it at 37 degrees for 1 hour.
Transformation is a process to maintain and propagate DNA sequences incorporated into plasmids in bacterial cells. You may observe many issues when analyzing the transformed colonies, such as few or no transformants, many colonies with no transforming DNA, etc. This section discusses common problems in bacterial transformation and suggestions on how to solve them.
Few or no transformants
●One of the important considerations is transformation efficiency. There might be suboptimal transformation efficiency.
○The transformation efficiency of competent cells in colony forms units per microgram of DNA (CFU/µg), generally ranging from 1 x 106 to 1 x 109 CFU/µg. Optimizing transformation efficiencies can greatly improve the likelihood of obtaining the desired clones.
■To ensure good transformation efficiencies, please be sure to store them at –70°C with minimal freeze-thawing and be more careful to handle them, avoid vortexing.
■Follow the transformation protocol and parameters recommended for the selected competent cells.
■Trying electroporation overheat shock for better efficiency with challenging DNA.
■Make sure the properties of the selected cells like transformation efficiency and genotype are appropriate for DNA transformation and intended applications.
■It is better to include a positive control in the transformation to verify the competence and transformation efficiency of the prepared cells.
[1] https://www.thermofisher.com/ca/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-troubleshooting.html
[2] https://www.thermofisher.com/ca/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/restriction-enzymes/restriction-enzyme-key-considerations.html
[3] https://www.thermofisher.com/ca/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/cloning/cloning-troubleshooting-guide.html
[4] https://www.thermofisher.com/ca/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-troubleshooting-guide.html
Contribution
In order to provide useful support and contribution to future iGEM, we have carried out the following two aspects of work.
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1. New Data
Ojective
Methods
Results
Conclusion
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2.Troubleshooting
PCR
Restriction Enzyme Digestion
Ligation
Transformation
- Reference