Team:XH-China/Notebook
Notebook
DAY1 7.31
After we got the plasmids, which was divided into two types. The first is carried by E. coli A, which is not good at expressing proteins. The second is a freeze-dried powder plasmid. Then we soak the second plasmid in saline, and we apply the second plasmid directly. Plasmids were placed in e. coli B and amplified on a medium plate. More importantly, we retained the first class plasmids embedded in E. coli A to prevent exhaustion.
DAY2 8.4
The plasmid was extracted, and then double-enzyme digestion was followed by agar-gel electrophoresis (Nucleic acid electrophoresis). We need to test whether the plasmid that had been introduced into E. coli is containing the target gene, the experimental result was that the target gene was included. After three hours of IPTG induction, protein electrophoresis was carried out, but no protein was found to be expressed.
DAY3 8.5
The reason is that the plasmids produced by the previous company did not contain his-tag and ampicillin resistant genes, which means that the vector plasmids containing the target genes sent by the company were not the vector plasmids that was required to be designed.So we're going to do a double digestion and move the target gene to a new plasmid that contains the HIS-tag and the ampicillin resistant gene.
DAY4 8.6
Double enzyme digestion of plasmids containing the target gene, two bands to determine that the target gene has been cut, and then expansion of e. coli containing the vector plasmids.
DAY 5 8.7
We extracted vector plasmids from the amplified Escherichia coli, configured with low melting point agar-gel electrophoresis, and used the cut plasmids containing the target gene to run the gel. After running the gel, the gel containing the target gene was directly cut and the target gene was recovered.
DAY6 8.10
The target gene was recovered last Friday, but the band was very thin. After the plasmid concentration was measured, it was found that the plasmid concentration was low and could not be directly connected to the carrier plasmid.
DAY7 8.11
Configure a low-melting agar-agar gel, run it with the target gene, then cut off the gel that contains the target gene, and this time recycle the target gene by putting it in a recycle column to increase its concentration.
DAY8 8.12
Even so, the concentration of the target gene collected was still low, so this morning both the vector plasmid and the plasmid with the target gene were double-digested, paired with low melting point agarose gel, and given an increased dose in the card slot, using the two plasmids mentioned above. It turned out to be no good, double digestion did not cut the vector plasmid and plasmid with the target gene. Then, because the plasmid with the target gene had run out, the plasmid was extracted again.
DAY9 8.13
Because the vector plasmid was also used up, we created the new competent cells (E. coli), the vector plasmid was converted, coated on a medium containing ampicillin, and one of the mediums was used as a negative control
DAY10 8.14
Double enzyme digestion and nucleic acid electrophoresis were performed on the target plasmid extracted previously, but it was found that the cutting was not successful, because the cutting time was too short, so a single colony of bacteria was selected from the positive culture medium.
DAY11 8.15
Ran electrophoresis of the target plasmid after the double enzyme digestion yesterday, found that the double enzyme digestion yesterday was successful, then extracted the vector plasmid amplified last night, and used the extracted vector plasmid to run gel in the afternoon, observed the concentration, and found that the concentration was high enough. At the same time, the target gene was cut into agar-agar gel with low melting point, and the target gene was directly cut into glue to recover. Then the target gene was connected with the extracted vector plasmid by double enzyme digestion.
DAY12 8.18
The attached plasmids were transformed into E. coli and a number of single colonies were selected from the transformed plate and coated on the medium.
DAY13 8.19
Single colonies were selected on the culture medium overnight.
DAY14 8.20
The plasmid was extracted from the overnight shaking solution and verified by double enzyme digestion. Two bands were found, indicating that the target gene was successfully connected to the carrier plasmid. The plasmids were then extracted and transformed into DH5 (amplified E. coli), coated on culture medium, and transformed into Rosetta (e. coli for expression) and coated on culture medium.
DAY15 8.21
Only one culture medium was found to have grown bacteria. However, because it was not labeled well, there was no way to tell whether the fungus on the culture medium was DH5 or Rosetta. So we first pick a single colony on the one with the bacteria and shake two tubes of bacteria.
DAY16 8.22
One of the two tubes of shaken bacteria was added with inducer IPTG, and the other tube was left untreated. After centrifugation, the concentration of bacteria was increased, and then the two tubes of bacteria liquid were subjected to electrophoresis.
DAY 17 8.24
After electrophoresis, it was found that the image was not displayed, so the bacteria were identified as DH5. After recoating Rosetta with medium, single colony shake bacteria were selected.
DAY18 8.25
ITPG inducer was added to Rosetta overnight bacterial solution to induce expression, and the plasmid was extracted by run-protein electrophoresis three hours after induction.
DAY19 8.26
The electrophoretic image showed that it was not expressed. The reason may be that ITPG was too old to use, so IPTG was reconfigured and the single colony was selected and put into the 1L culture bottle to cultivate Rosetta.
DAY20 8.27
Inducers are added to induce Rosetta in a large culture flask and run to see if it expresses the protein.
DAY21 8.28
Western blot of image signal is too weak, said there was no protein expression, so do the error analysis, plasmid extraction on Tuesday we came out and control plasmid DNA electrophoresis, found that we don't have image of plasmid, plasmid were not transformed into e. coli, e. coli but this is still in the AMP (ampicillin) of medium long out, even if not one of them contains AMP resistance genes. The reason may be due to AMP failure or other reasons, so a new tube of Rosetta bacteria that has been identified for plasmid introduction was found.
DAY22 8.29
The shaken Rosetta was poured into a 1L large culture flask, followed by IPTG induction for 3 hours, and protein electrophoresis was performed to determine whether the protein was successfully expressed. Fortunately, it worked.
As shown in the figure, the expression of keratin in samples was induced by IPTG
and detected by SDS-PAGE and Western-blot for 3 h after induction.
Swimlane 1 is the electrophoretic band of the uninduced sample, and swimlane 2 is the induced expression sample band.Induced bands were detected in Wb test, and induced keratin could be expressed in resuspended bacterial fluid.
DAY23 8.31
As shown in the figure, after inoculation of a large amount of keratin induced expression with the bacterial solution with a small amount of expression, the bacteria were collected by centrifugation, then ultrasonic crushing was performed, supernatant and precipitation were collected by centrifugation, and the precipitation was dissolved with 8M- urea buffer, and sampled and electrophoretic respectively.
Swimlane 1 is the electrophoresis band of supernatant sample, and swim lane 2 is the electrophoresis band of precipitate dissolved sample.
There were bands in both clearing and precipitation at the same size as keratin, so it was impossible to determine whether it was keratin or not, and western-blot detection was needed.
DAY24 9.1
As shown in the figure, after a large amount of expression, the bacteria were collected by centrifugation, followed by ultrasonic crushing, supernatant and precipitation were collected by centrifugation, and the precipitation was dissolved with 8M- urea buffer, respectively sampled and electrophoresis, and western-blot detection was carried out
Swimlane 1 is the electrophoresis band of supernatant sample, and swim lane 2 is the electrophoresis band of precipitate dissolved sample.
It can be seen from the figure that keratin is mainly expressed as soluble protein.
DAY25 9.2
As shown in the figure, ultrasonic broken crude protein supernatant samples were taken for purification
Lane 1-3 is 20mM imidazole phosphate buffer to wash the mixed samples, adding in 5 times, each time adding 5mL.
Lane 4-7 is the sample eluted with 250mM imidazole phosphate buffer, which is added in 4 times and 5mL each time.
Keratin can be purified by ni-NTA preloaded with gravity, and the purified protein band is relatively single, the sample purity is high, but the yield is low.
DAY26 9.3
The activity of purified keratinase was measured by Sigma non-specific protease activity detection method, and the standard curve of content micromole and absorbance value OD660 was established. After calculation, the activity of the synthesized keratinase was calculated as 4.24 units /ml/ min.