Team:CIEI-BJ/Notebook
January 30th – February 10th ，2018
Our team was basically founded. The team members participated in a 12-day training on fundamental theory and basic experimental skills and started our brainstorming on the project.
May 20th ，2018 Group Meeting
We came out with some ideas about our research direction. After several rounds of discussions, we decided to build up a system to degrade aflatoxin.
May 27th ，2018 Group Meeting
We decided on the exact functions of our system and all of us began to search for background information before finalizing our summary.
July 13th -29th ，2018
Most of team members gathered at the University of Science & Technology Beijing for competition preparation.
July -22nd ，2018 Group Meeting
All the team members and advisors had a meeting to discuss on the focus of the project. A more detailed schedule was made.
August 26th ，2018 Group Meeting
The focus of our project was further clarified after several discussions. Each group of the team reported on their progress. And a specified time plan was agreed on.
September 2nd ，2018 Group Meeting
This time, we mainly focused on improving human practice part and pushed it forward. Our questionnaires and funding proposal were finished online.
October 3rd - 4th ，2018
All team members gathered to work on the poster, wiki and improving our presentation skill. The poster and PowerPoint slides for the final presentation was almost ready.
March 15th, 2018
ADTZ-SC sequence was synthesized and integrated into vector pUC57
March 17th-18th, 2018
The gene was added digested site through PCR
March 24th-25th, 2018
Cut and purify the PCR product (described above) and vector pYES2
April 7th, 2018
The digested product of ADTZ-SC was integrated into vector pYES2
April 8th, 2018
The ligation product was transformed competent cells of E.coli, and checked by PCR
April 21st-22nd, 2018
Isolate the plasmid from positive colony
May 5th-6th, 2018
ADTZ-SC-pYES2 plasmid was used to transform yeast strain INVSc1
May 12th -13th, 2018
Check recombinant yeast colony by PCR
June 2nd-3rd, 2018
Yeast cell culture
June 9th-10th, 2018
Extract proteins from the yeast
June 23rd-30th, 2018
Examine if yeast can degrade aflatoxin
July 7th-15th, 2018
pYES2-ADTZ total protein enzyme activity analysis in vitro
Soluble expression of ADTZ in E.coli and the degradation of aflatoxin B1 by ADTZ
September 23rd , 2018
Design primers for pMAL-c5x- ADTZ construction
September 24th , 2018
Amplify ADTZ fragment and linearized pMAL-c5x fragment by PCR
Purify PCR product.
September 25th , 2018
In-Fusion
Transformation
September 26th , 2018
Colony PCR
September 27th , 2018
Miniprep plasmid pMAL-c5x-ADTZ
Sequence pMAL-c5x-ADTZ
September 29th , 2018
Prepare E.coli BL21 Gold DE(3) competent cells
Transform pMAL-c5x-ADTZ into E.coli BL21 Gold DE(3)
September 30th , 2018
Inoculate E.coli BL21 Gold DE(3) containing pMAL-c5x-ADTZ into 3mL LB, culture overnight at 37 oC and 200rpm
October 1st, 2018
The overnight culture of the bacteria was inoculated into LB broth containing 2g/L glucose and 100mg/L ampicillin. After 2h culture at 37 oC and 200rpm, recombinant protein production was induced by addition of 0.3 mM IPTG at 20oC and 200rpm for 24h.
October 2nd, 2018
The expressed cells were collected by centrifugation and the pellet was resuspended in H2O about an OD600 of 30. The cell was disrupted by sonication. SDS-PAGE analyzed the expressed MBP-ADTZ fusion protein.
October 3rd, 2018
Perform the degradation reaction of aflatoxin B1 and prepare the samples for HPLC.
October 4th, 2018
HPLC analysis
August 10th, 2018
The design of the experiment：
August 20th, 2018
Construction of the vectors:
| pGADT7-pGal1-BacC; |
| pGADT7-pGal1-ADTZ; |
| pGADT7-pGal1-MNP; |
| pGADT7-pGal1-MSMEG5998; |
| pGBKT7-pGal1-Eyfp; |
August 21st, 2018
Screen colonies using carboxybenzyl plates and verify the colonies through PCR with the 2F/2R primer. The gene length is 442bp. Then choos positive colonies to sequencing using primer t7-pro / 3’AD.
August 22nd, 2018
Design the primer ADpGAL1-F/R and BDpGAL1-F/R to amplify ADter and promoter GAL1 which will construct the downstream of ScFv1/2 in order to insert more candidate genes.
August 23rd, 2018
Screen colonies using carboxybenzyl plates and verify the colonies through PCR with the t7-pro / 3’AD primer. The gene length is 1700bp. The sequencing results of positive clones are right.
August 24th-September 3rd, 2018
Transform
| pADT7-ScFv1; |
| pADT7-ScFv1-BacC; |
| pADT7-ScFv1-MNP; |
| pADT7-ScFv1-ADTZ;; |
| pADT7-ScFv1-MSMEG5998 |
to yeast strain AH109 with pBKT7-ScFv2 and pBKT7-ScFv2-eYFP, separately. After transformed three days (8.24 to 8.27), we got the result of pADT7-ScFv1&pBKT7-ScFv2 and pADT7-ScFv1-BacC & pBKT7-ScFv2:
September 3rd, 2018
The yeast cells transformed with two plasmids were cultured in SD-His media with no Aflatoxin B1 (control) or 100 ug/L (100 ppb) AFT B1. According to our design, the presence of AFT B1 should facilitate the association of Gal4 AD and BD, thus driving the expression of His3 protein under the endogenous Gal1 promoter, and enabling yeast growth in SD-His media. The yeast growth (OD 600) was measured every 2 hour for 14 hours on end, and the growth curve clearly showed that, compared to control, the presence of AFT B1 significantly promoted the growth of the yeast. Therefore, the results shows the similar changes with AD-ScFv1 (BBa_K2247008) and BD-ScFv2 (BBa_K2247009). Besides this, the candidate genes which may degrade AFB1 shows very hard to growth in SD-His media. Whether these genes are really working, should be further confirmed by more experiments.
September 7th, 2018
The eYFP is working under the AFB1 existing conditions in the yeast cells.
Western bolt to confirm the EYFP protein can be induced in the presence of AFT.