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Team:NEAU-Harbin/notebook.html
From 2014.igem.org
We learnt and planed the preliminary experiment, and prepared some of the materials needed during the experiment, such as LB medium. In this week, we got familiar with the operation of some basic equipment and our future experiment. In the meantime, we received four strains of E.coli which contain pSZH, pAN7-1, pSZH-G and pSZH with GlaA3, and carried rejuvenation and preservation out for them. We got the overnight suspension culture and extracted the plasmid from these four kinds of E.coli.
Week 2We received the parts library from iGEM HQ , dissolved the plasmids which containing genes of Crtw-rbs, Crtz-rbs, CrtY, CrtB, CrtI-rbs, CrtB-rbs, CP, cjBlue, and RFP with ddH2O and removed them out to transform them into DH5α competent cells.
Week 3We attempted to eliminate the Not I restriction site. We used Not I restriction enzyme to digest the plasmid pSZH (37℃ overnight ), and recovered by using centrifugal column method . The linear DNA recovered by us was reinforced and extended at 72℃ in the PCR instrument for 5min . We recovered the plasmid again ,but unfortunately , we failed because of the low percent recovery . The fragment is too large and the recovery kit we used has a limited scope , which may lead to the low efficiency of recovery .But the good news is that the transformation of 9 genes last week was successful and the 9 kinds of plasmids containing the genes were extracted .Since the pigment metabolism genes only can be used later , firstly, we digested the 3 kinds of fluorescent protein genes ( CP, cjBlue , RFP ) by Xba I and Not I (We added the Xba I into the solution firstly to react 2h and then we added Not I to react another 2h). Agarose Gel Electrophoresis showed that the digest reaction was complete .
Week 4We get a plasmid containing the HPH and GlaA and primer (including Linker) of GlaA3, HPH, and ultraviolet excitated GFP, cjBlue, RFP, BFP, using PrimeStar catalyze PCR, and the product was electrophoresed in agarose -TAE gel, tapping and recycle the target segments. Unfortunately , because of the error of design , we didn't get the right fragment . Then we use Taq to add “A” to the recycling product in PCR machine (72℃ for 1h), and recycle the product in liquid (spin column method) and mix them with T vector , linking at 4℃ overnight . The product was transformed to DH5α competent cell and vaccinated on Amp+ LB plate culture overnight . In addition , we tried to knockout the NotI restriction site. We changed the recovery kit, but the effect is still not good .
Week 5Luckily , strains with the T vector containing the 5 genes transformed last week grew well . We inoculated the 5 kinds of single colony to liquid Amp+ LB medium, shaking cultured overnight at 37℃ 200rpm and extracted the plasmids of them, which was successful . We redesigned primers , and amplificated cjBlue by PCR . Unfortunately , we failed again . We consulted instructors , redesigned primers .
Week 6We got primers of cjBlue ,and successfully amplificated the cjBlue segment . We linked it to T vector and transformed it to DH5α competent cell. Single colonies were inoculated to liquid LB medium and plasmids were extracted. This week , we knockouted the NotI restriction site again by a recycling kit with DB . The strips in agarose-TAE gel electrophoresis had long tails that is called Comet strip, which indicated that the vector was degraded . But there was a clear strip in the position , so we tried to operate the blunt-end ligation by using T4 ligase (We divided the reaction system into 4 parts . Part I had 1μL plasmids. Part II had 2μL plasmids. Part III had 4μL plasmids.)
Week 7The pSZH plasmid was transformed into DH5α. Luckily , it grew well on Km+ solid LB medium , and we got many colonies . We choose 10 colonies , to grow them in liquid Km+ LB medium , and extracted their plasmid . We were surprised to find that all the plasmids were about 3000bp. We suspected that the plasmids were degraded but the segment of Km+ resistance were kept with the losing of other segments. In a word, the knockout of NotI was failed again . Besides this, we operated double digestion of N-HPH (NcoI and EcoRI), F-cjBlue (EcoRI and BamHI) , GlaA3 (BglIIand XbaI) , pAN7-1 (NcoI and BamHI) , CP (ApaI and HindIII) and pSZH-G (ApaI and HindIII) in 20 μL systems . Agarose-TAE gel electrophoresis showed that, the digestion was thorough and effective except CP and pSZH-G. So we digested N-HPH , cj-Blue, GlaA3 and pAN7-1 in 100μL reaction systems, and recycled them in agarose-TAE gel. The recycling effect of pAN7-1 is low. We tried to link pAN7-1, F-cjBlue and N-HPH . We transformed the product into DH5α, and spreaded on solid Amp+ LB medium .
Week 8Growth of the E.coli transformed with pAN7-1 vector is not well. We analyzed the reason and find that the recycling effect of pAN7-1 was very low which makes the connection is difficult. This week , we digested N-HPH (NcoI and EcoRI), F-cjBlue (EcoRI and BamHI) , GlaA3 (BglIIand XbaI) , pAN7-1 (NcoI and BamHI) , CP (ApaI and HindIII) and pSZH-G (ApaI and HindIII) again , and recycled them in agarose-TAE gel. Unfortunately , the recycling effect was not good too. For the knockout of NotI restriction site in pSZH , we used ethanol precipitation recycling instead of spin column method, which got a good effect. Then blunt ends of the product recovered by us had been connected together for 3 days.
Week 9This week we have a temporary break and analyze errors occurred before.
Week 10We extracted the pSZH plasmid whose blunt ends have been connected in Week 8 , and used ApaI and NotI to operate double digestion identification . At the same time, we restreaked rejuvenation all strains used before , and extracted their plasmid standing by. We digested N-HPH (NcoI, EcoRI), F-cjBlue (EcoRI and BamHI), ultraviolet excited GFP (EcoRI, BamHI), GlaA3 (BglII, XbaI), and pAN7-1 (NcoI, BamHI) , each in two 100μL system, and recycled them in Agarose-TAE gel. Because of the discussion about the reason of inefficient recycling last week , we improved the operation of gel tapping and recycling , which makes the efficiency was greatly improved except the strip of pAN7-1 vector was still dark . We mixed N-HPH, F-cjBlue, pAN7-1 and N-HPH, GFP, pAN7-1, and added T4 ligase into the reaction system and let them react for 48h.
Week 11The two ligation product systems in last week were transformed into DH5α and inoculated on each two Amp+ solid LB mediums to be cultured overnight . There were two single colonies on the two mediums of F-cjBlue , but there was nothing on the one of GFP. We used primers of cjBlue to operate PCR for the 4 colonies , and the result indicated that they were all positive . Then we inoculated them into Amp+ liquid LB medium and cultured in 37℃ for 12h. Two bottles of broth are green and there was no cjBlue product in another two bottles. We extracted plasmids from them but failed in the green broth . We used EcoRI and BamHI to digest them , showing positive and the size of fragment is same with cjBlue , which indicated that N-HPH and cjBlue are connected to pAN7-1 successfully . At the same time , we inoculated pSZH-G into Km+ liquid LB medium and CP into Amp+ liquid LB medium . Plasmids were extracted , and digested by ApaI and HindIII in 100μL reaction systems. We recycled the CP product from Agarose-TAE gel and pSZH-G from liquid. Because of inefficient extraction of pSZH-G , the concentration of recycling product is not enough , but we recycled CP successfully whose strip in Agarose-TAE gel electrophoresis is bright . So we tried to connect them (T4 ligase, 48h) . This week we tried to knockout NotI again, using alcohol precipitation recycling pSZH digested . Because of operational problems , we failed again .
Week 12We transformed the pSZH-G and CP into DH5α, and inoculated on Km+ solid LB medium , which grew well . Then we selected 20 colonies to operate PCR , they were all positive . A bottle of broth was purple and we suspected that there was T vector mixed in it. Other colonies were cultured and plasmids were extracted and restriction identification was negative . So this time was failed again . But we find that chloramphenicol can improve the copy number of large vector pSZH-G, so we tried to optimize the bacterial culture solution , i.e. , inoculate noon and add chloramphenicol into it (final concentration is 60μg / mL), and extract plasmids next day . After many lessons learned , this method is excellent for the extraction of large vectors. In addition , we also PCR the crtE, crtB, crtI, crtY, crtZ five genes. Restriction enzyme digestion showed that the four except crtE all cut out of the target strip . We recycled them and add Adenine deoxynucleotide to its end for the connection with T vector and transformed into DH5α . However , crtE had 3 strips which was dark . So we operated PCR again , and repeated the operation again . At the same time , this week we made 8 tubes of 100μL reaction systems for finding reason of the failure of NotI knockout . Four of them were recycled by using spin column method and another four used alcohol precipitation recycling method . We compared with the two methods , result indicated that alcohol precipitation recycling method had a higher efficiency than another one under the circumstance that all operation are right . We used PCR instrument to fill the cohesive ends of recycling product (72 ℃, 5min), and recovered by using ethanol precipitation method .
Week 13Plasmids of pSZH-G , CP and RFP were extracted and digested again ( RFP : ApaI and ClaI ) . We found that pSZH-G and CP were digested thoroughly , but there were not target strip shown in electrophoresis . We analyzed the reason and found that methylation makes ClaI can not digest its restriction sites. So we transformed RFP(in the T vector) into Trans110 competent cell, and got the strain containing RFP genes that was not methylated. We extracted plasmids from it and digested , which was more efficient . CP and RFP were recycled from Agarose-TAE gel and pSZH-G was recycled from liquid. Unfortunately , only CP got a nice recycling. The failure of RFP was because of operation errors, and pSZH-G was because of its large size which can cause a huge loss in spin columns recycling method. We recycled the pSZH whose cohesive ends were filled last week , and failed .
Week 14We inoculated RFP strains and extracted the plasmid which was digested and recycled . We got a good efficiency in recycling this time . At the same time , we digested and recycled pSZH-G , and increased the size of reaction system , which makes the plasmid recycled more than before . We operated ligation (T4 , 48h), and transformed product into DH5α competent cell which was cultured on solid Km+ LB medium (37℃) . For the knockout of NotI in pSZH-xynB , we designed an adapter which is 14bp to connect cohesive ends and avoid the embarrassment of low recycling efficiency . We mixed the two parts of adapter and connected them under 70℃ in PCR instrument and cooled down at room temperature. Finally , product was cooled down in ice-bath for 5 min and took 3μL and 5μL for connection at 16℃ for 2 days. This week we digested pSZH-G(ApaI, HindIII) . Then we connected CP and pSZH-G and transformed into DH5α which was cultured on Km+ solid LB medium. We got 22 colonies totally , and selected 15 colonies randomly to operate XbaI and XhoI digestion identification . The strips cut down was around 4500bp, which had a large difference with 3000bp which is the theoretical value.
Week 15We obtained the correct pSZH-G plasmid and transformed it into DH5α competent cells to coating on Km+ solid LB medium . Single colonies were inoculated to liquid Km+ LB medium and plasmid from them were extracted . Digested them as usual and divided them into two 100μL reaction systems . One was recycled by using glass milk method and another one used opinion column method . The result indicated that , glass milk method has a higher efficiency in the recycling of large vector . We connected product with CP recycled before (pSZH-G , 5μL. CP , 3μL. T4 ligase) for 48h , and transformed into DH5α competent cells and coat on Km+ solid LB medium to culture overnight and got 14 colonies . We operated colony PCR , only 1 colony was positive and we suspected that electrophoresis roads of other colonies were jammed . We cultured the positive colony to extract its plasmid and operated digesting identification (XbaI, HindIII) . Six colonies were suspected positive . We chose No.1 and No.13 whose target strips were bright to culture in Km+ liquid LB medium and extracted plasmids . We finished the improvement of pAN7-1 and connected it to pSZh-xynB digested by XhoI and NheI . Unfortunately , there were many restriction sites on it, and cohesive ends formed by NheI and XbaI can be linked together , which can delete the restriction site . So we cannot use them to identify the plasmid. We PCRed plasmid and digested it by KpnI for identification , which indicated that the plasmid was positive.
Week 16We digested the plasmid extracted last week by XbaI and XhoI, there many strips in Agarose-TAE gel electrophoresis. We asked many people in the lab , and finally found that XbaI was polluted by other restriction enzyme . After many tests, we produced Agrobacterium competent cells to transform No.13 and No.1 plasmids into them and coated the mixture on six Rif+ solid YEB mediums(30℃).
Week 17Agrobacterium on six YEB plates grew well. We PCRed their colonies (CPs, CPas) and they were all positive , which means plasmids are all transformed into them . So we inoculated one colony into liquid Rif+ YEB medium, shaking cultured and took some broth to mix with Aspergilus Niger and co-cultured 3 days on solid PDA mediums(added AS) which covered by glass papers .
Week 18We moved the glass papers covering on PDA plates which were used in the co-culturing to PDA plates containing cefotaxime and hygromycin and cultured 1 day. We removed glass papers from PDA plates and put them in 30 ℃ incubator for culturing.
