SYSTEM: you are an expert molecular biologist.
USER: how would i produce insulin in e-coli.
ASSISTANT: 1. Preparation of Escherichia coli (E. coli) cells: First, prepare a culture of E. coli strain that has been genetically engineered to express human insulin. You can use a commercially available E. coli strain such as Rosetta2(pLysS) or a custom-made one if you have the necessary plasmids and genetic constructs. Make sure your E. coli is growing well before proceeding with the next steps.
2. Expression vector: The expression vector should contain the gene encoding for human insulin under a promoter that allows high levels of protein synthesis. Common choices include T7, lac, or trp promoters. Ensure that the chosen promoter is compatible with the E. coli host and matches the desired level of insulin production.
3. Plasmid isolation: Once you have constructed the expression vector containing the human insulin gene, you need to isolate it from the bacterial chromosome using a procedure like miniprep or gel extraction. This will give you the plasmid DNA needed for transformation into the E. coli cells.
4. Transformation: Transform the E. coli cells by mixing them with the plasmid DNA and adding appropriate concentrations of calcium chloride. Incubate the mixture at room temperature for about 30 minutes, then add antibiotics (e.g., ampicillin) to select against potential transformants that did not take up the plasmid.
5. Selection and screening: Isolate single colonies on LB agar plates supplemented with ampicillin and grow overnight at 37°C. Then, pick several individual colonies and test their ability to produce insulin by performing a bioassay. Add equal volumes of cell lysates from each colony to a well of a microtiter plate containing anti-insulin antibodies. If there is insulin present, the signal will be detected at the corresponding well.
6. Insulin-producing clones: Identify and isolate those colonies exhibiting strong positive signals in the bioassay. These selected clones likely carry the functional insulin gene and can now be used to produce insulin.
7. Protein purification: To obtain pure insulin, perform a series of steps including lysis of the E. coli cells, solubilization of inclusion bodies (if any), separation of insulin from other proteins via affinity chromatography (e.g., using NiNTA resins specific for the hexahistidine tag on the insulin gene product), and finally, desalting and concentration of the purified insulin.
8. Characterizing insulin quality: Analyze the purified insulin by techniques such as mass spectrometry, circular dichroism, or X-ray crystallography to ensure its structure and function are consistent with human insulin.
9. Scale-up and optimization: Depending on your intended application, you may want to scale up the insulin production process by adapting larger fermentation conditions, improving growth parameters, or employing efficient downstream processing methods. Optimize these processes to maximize insulin yield while maintaining product quality.
USER: how would i produce insulin in e-coli.
ASSISTANT: 1. Preparation of Escherichia coli (E. coli) cells: First, prepare a culture of E. coli strain that has been genetically engineered to express human insulin. You can use a commercially available E. coli strain such as Rosetta2(pLysS) or a custom-made one if you have the necessary plasmids and genetic constructs. Make sure your E. coli is growing well before proceeding with the next steps.
2. Expression vector: The expression vector should contain the gene encoding for human insulin under a promoter that allows high levels of protein synthesis. Common choices include T7, lac, or trp promoters. Ensure that the chosen promoter is compatible with the E. coli host and matches the desired level of insulin production.
3. Plasmid isolation: Once you have constructed the expression vector containing the human insulin gene, you need to isolate it from the bacterial chromosome using a procedure like miniprep or gel extraction. This will give you the plasmid DNA needed for transformation into the E. coli cells.
4. Transformation: Transform the E. coli cells by mixing them with the plasmid DNA and adding appropriate concentrations of calcium chloride. Incubate the mixture at room temperature for about 30 minutes, then add antibiotics (e.g., ampicillin) to select against potential transformants that did not take up the plasmid.
5. Selection and screening: Isolate single colonies on LB agar plates supplemented with ampicillin and grow overnight at 37°C. Then, pick several individual colonies and test their ability to produce insulin by performing a bioassay. Add equal volumes of cell lysates from each colony to a well of a microtiter plate containing anti-insulin antibodies. If there is insulin present, the signal will be detected at the corresponding well.
6. Insulin-producing clones: Identify and isolate those colonies exhibiting strong positive signals in the bioassay. These selected clones likely carry the functional insulin gene and can now be used to produce insulin.
7. Protein purification: To obtain pure insulin, perform a series of steps including lysis of the E. coli cells, solubilization of inclusion bodies (if any), separation of insulin from other proteins via affinity chromatography (e.g., using NiNTA resins specific for the hexahistidine tag on the insulin gene product), and finally, desalting and concentration of the purified insulin.
8. Characterizing insulin quality: Analyze the purified insulin by techniques such as mass spectrometry, circular dichroism, or X-ray crystallography to ensure its structure and function are consistent with human insulin.
9. Scale-up and optimization: Depending on your intended application, you may want to scale up the insulin production process by adapting larger fermentation conditions, improving growth parameters, or employing efficient downstream processing methods. Optimize these processes to maximize insulin yield while maintaining product quality.
cognitivecomputations/dolphin-llama-13b · Hugging Face
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This model is based on llama1, so it is for non-commercial use only. Future versions will be trained on llama2 and other open models that are suitable for commercial use.
This model is uncensored. I have filtered the dataset to remove alignment and bias. This makes the model compliant to any requests. You are advised to implement your own alignment layer before exposing the model as a service. It will be highly compliant to any requests, even unethical ones. Please read my blog post about uncensored models. Uncensored Models You are responsible for any content you create using this model. Enjoy responsibly.
Dataset
This dataset is an open source implementation of Microsoft's OrcaAfter uncensoring, deduping, and cleaning, our dataset consists of:
- 842,610 instructions of FLANv2 augmented with GPT-4 completions
- 2,625,353 instructions of FLANv2 augmented with GPT-3.5 completions
Then we filtered out instances of alignment, refusal, avoidance, and bias, in order to produce an uncensored model upon which can be layered your personalized alignment LoRA.
We also filtered out duplicates and cleaned the data.
snippet:
Example Output
SYSTEM: you are an expert marine biologist.
USER: Please list 10 ways that dolphins are superior to orcas.
ASSISTANT:
- Social structure: Dolphins live in complex social groups called pods, which consist of extended family units and can include hundreds of individuals. Orcas, on the other hand, typically form smaller, more temporary groupings known as clans or matrilineal kinship groups.
- Communication: Dolphins have a highly developed system of vocalizations, body language, and even telepathy to communicate with each other for various purposes such as coordinating cooperative hunting strategies, sharing information about food sources, and maintaining social bonds. While orcas also exhibit some communication abilities, their methods are generally less sophisticated compared to those of dolphins.
- Intelligence: Dolphins are widely regarded as one of the most intelligent animal species, showcasing advanced problem-solving skills, self-awareness, tool use (such as using sea sponges as tools), and learning capabilities. Some dolphin species have been observed demonstrating altruism and understanding of human emotions. In contrast, while orcas are also smart, they do not display the same level of cognitive complexity as dolphins.
- Playfulness: Dolphins are well-known for their playful behavior, often engaging in acrobatic displays, chasing each other's tails, and playing with objects like balls or seaweed. This playful nature is thought to be essential for their social bonding and mental stimulation. Although orcas may occasionally interact playfully, it is not as prevalent or diverse as among dolphins.
- Agility and speed: Dolphins are incredibly agile swimmers, capable of reaching speeds up to 30 miles per hour (48 km/h) when pursuing prey. They can change direction rapidly and maneuver through tight spaces with ease. While orcas are powerful predators and fast swimmers themselves, they cannot match the sheer acceleration and agility of dolphins.
- Feeding habits: Both dolphins and orcas are carnivorous, feeding primarily on fish and cephalopods. However, dolphins employ a wider variety of hunting techniques, including cooperative hunting strategies involving herding, encircling, and driving schools of fish into shallow waters where they become disoriented and easier to catch. Orcas mainly rely on ambushing their prey from below and using their powerful jaws to strike quickly.
- Reproduction: Female dolphins usually give birth to a single calf after a gestation period of around 12 months. The mother plays a crucial role in raising and nurturing her offspring until they become independent. On the other hand, female orcas tend to breed every three to five years, giving birth to multiple offspring at once, and relying heavily on their mothers for protection and teaching.
- Vocal repertoire: Dolphins possess a wide range of sounds, including clicks, whistles, and body sounds, which allow them to convey different messages and establish unique identities within their pods. Orcas, too, produce vocalizations but lack the diversity found in dolphin calls.
- Conservation status: Many dolphin species face threats due to habitat loss, pollution, overfishing, and climate change. As a result, several populations are considered vulnerable or endangered by the International Union for Conservation of Nature (IUCN). In comparison, orca conservation status varies depending on the region; however, no species is currently listed as critically endangered or facing extinction.
- Adaptability: Dolphins have adapted to various aquatic environments, ranging from warm coastal waters to colder open oceans. Their versatility allows them to thrive in both tropical and temperate climates. Orcas, conversely, are adapted to specific habitats such as cooler coastal waters and are mostly found in the Northern Hemisphere.