Team:GZHS-United/Notebook

Notebook

Date objection result Analysis Solution
7-10 1. Extract the genomic DNA of Bt and Bs. 1. Only the genomic DNA of Bs were obtained. The Bt haven’t been actived. Culture Bt in LB to active it.
7-11 1. Extract the genomic DNA of Bt.
2. Acquire mtx gene form genomic DNA of Bs by PCR. 		1. DNA electrophoresis showed that the strip appeared at 150bp.
2. A peal DNA strip in agarose gel electrophoresis were observed. 		1. The fragment we extracted were RNA. 1. Use a more gentle method to broke the Gram-negative bacterium cell wall.
2. Optimize the PCR condition and try to use the diluted production as template.
7-12 1. Extract the genomic DNA of Bt.
2. Amplification of mtx1 gene. 		1. The genomic DNA of Bt were successfully obtained.

2. The mtx1 gene was successfully obtained. 		Boiling water-bath is a good method to broke the bacterium cell wall. /
7-13 1. Acquire Cry4Ba gene form genomic DNA of Bt by PCR.
2. Purification the PCR product of mtx1 .
3. Construction of cloning vector for direct ligation with mtx1’s purified product .
4. Transform the plasmid into E.coli. 		1. The Cry4Ba gene was successfully obtained.

2. A series of Experiment were performed successfully. 		/ /
7-14 1. Use colony PCR to select positive colonies among white colonies that we got from the blue-white selection. We set three groups of colonies (A,B and C)
A: mtx1
B: Cry4Ba (primers with enzyme digestion sets)
C: Cry4Ba (primers without digestion sets)
Each group selected 14 colonies. Operation was conducted in super-clean bench. So as to preserve the bacterial of colonies, we took 42 centrifuge tubes and added 750uL of cultural medium in each tube. We mix the bacterial in the cultural medium before adding into the reaction system for PCR.
2. Make up new LB cultural medium (Cultural medium we used yesterday may not be reliable).
3. Spreading positive control (BBa_I20270) and negative control (BBa_R0040) on the plate so as to get transformant, each duplicated for two groups.
 Result of colony PCR.

According to this result, we selected one colony from group B, one colony from group C and two colonies from group A for sequencing.
The outcome of cry4Ba is really good, but we may need more test to figure out the best temperature for annealing. 		1. Be careful not to contaminate the samples during the operation on super-clean bench.
2, Be aware that once leave the super-clean bench, the operator has to extinguish the spirit lamp 		The whole team.
7-15 1. Transform BBa_I20270 and BBa_R0040 transformants into LB solution and culture in flasks.
2. Distribute the missions of human practice and wiki. 		No experimental outcome, but made many plans for future work. The whole team.
7-16 1. We received the result of sequencing for cry4Ba and Mtx1. According to the result, we have succeeded in assembling cry4Ba into the cloning vector but failed in Mtx1, so we have to redo the extraction of genome DNA in Bs.
2. Extract plasmid from cry4Ba transformant. 		The gel of genome and plasmid extraction:

Lane 1：Marker DL5000
Lane 2：Bs genome (failed)
Lane 3：Bs genome (failed)
Lane 4：Extraction of cloning vector with
cry4Ba (Low in cconcentration)
Lane 5：cry4Ba amplified by PCR (use the
extracted plasmid as template)
summary: Failed in extracting Bs genome again. The cloning vector actually assembled cry4Ba, but the result of gel is not clear due to the low concentration of plasmid. 		Note for plasmid extraction:
1, Ensure that the rotor of centrifuge is balanced before centrifuge.
2, Be aware of the time for reaction in each steps.
3，One of the important steps which can determine the outcome of the extraction is standing and demix. Be careful not to mix the precipitation of protein with DNA in the supernatant.
4, control the speed of centrifuge precisely. 		The whole team.
07-30 We got the eGFP sequences (BBa_K404316) from the iGEM registry and redesign nest-PCR primer. Figure 1 No control, try again Control-1: BL21，
Control-2:BL21+ cry4Ba
07-31 1. Explore the best ratio of fodder
2. Feed larva
3. Test toxicity of bacterias 		1. 0.0125g agar+1mL H2O+0.02g cat food
2. table1
3. table2-result
08-01 1. Redesign the experiment of toxicity
2. Fuse GFP and CRY4Ba and amplify mtx1 by pcr 		1. table3
2. Figure2

 2 Failed 2 You know nothing!
08-02 1.The fourth amplification of Cry4Ba-eGFP fused gene 2.Large scale production of Cry4Ba-eGFP fused gene
3.Cry4ba --eGFP Fragment cloning
 1.

2.
 We modified the PCR pattern to amplified Cry4Ba-eGFP and got the result shown as the figure on the right. It could be observed that there was a faint belt between 3000bp and 5000bp, we speculated that this was the Cry4Ba-eGFP fused gene.
08-03 1. Screening of Cry4Ba--eGFP Fragment Vector Carrier Positive Bacteria. We choose single bacteria as model of colony PCR. The normal primers are the M13F/M13R. And the sequence is shown in the right site. As the photo said, there are 3 kinds of consequences shown on the Electrophoretic gel, the Blank, the 1000+bop sequence and the empty vector(300bp)..
 Although we didn't get the goal size fragment by amplifying, which is the positive recombinant, for trying, we sent the A1 for testing and keep the empty model(A5,8,9,11etc.)
08-04 1. Sample A1 sequencing As the Sample A1 sequence is shown in the photo in the right sight. The consequence indicates that the inserted sequenceof coloring vector is a GFP sequence. The 5-terminal has a Cry4Ba-T1-F primer sequence. We also found that the last 4 free Nuclotides in the 5-terminal are the same with the 4 Nuclotides in the 5'-terminal of the 4BaGFP-T1-F.(GAAT) When being free in the 4BaGFP-GFP5-terminal(GFP carrying overlapping sequence), cry4Ba-T1-F and 4BaGFP-GFP5'-terminal match each other. Because it is much easier to amplify for the short sequence, the amounts of non-specific fragments produced by the system is much more than purpose fragment when the free 4BaGFP-GFP sequence in the 5'-terminal has a larger quantity. So we can always get the consequence about 750bp non-specific band if we conduct the PCR for overlapping extension.
 The sequencing results indicate that it will be failed to construct a fused protein vector when inserting a non-specific sequence in the coloring vector.
08-05 we got vectors from BL21 that had no positive band in colony PCR but can still grow, and we used Pstl to cut it. The result of enzyme cutting showed that the target gene failed to be inserted in the cloning vectors. We used the PCR primers: Cry4Ba-T1-F/4BaGFP-R to amplify Cry4Ba gene that has overlapping segment, and we used primers, 4BaGFP-F/GFP-R, to amplify the GFP with overlapping segment. After amplifying the the target genes, we purified them and prepared next PCR round.
Basing on the rebuilt Cry4Ba and EGFP, we changed the method and continued to do the fifth overlapping PCR. The result is on the left. CK contains same amount of Cry4Ba and eGFP. The result indicateed that, this time we got many segments that have same size as target genes form overlapping PCR. After Gel Extraction Purification, we started building the expression vectors and the cloning vectors at the same time in order to save time.
08-08 1、cry4Ba-eGFP screening of positive recombinant colony PCR. The result of bacterial colony show above, CK+is the template of cry4Ba segment, and use TY1644-W1F/ GFP-R primer for protein amplification，for positive contrast。The result show that，lysozymelike 1 is the n orientation positive recombinants.
 We choose to use lysozmelike 1 for expansion culture. Use for extract pEASY-Blunt-E1(cry4Ba-eGFP) recombinant plasmid.
08-09 Record was lost. Record was lost. Record was lost. Record was lost.
08-10 1. Extract pEASY-Blunt-E1(cry4Ba-eGFP) from E.coli DH5α
2. Transform pEASY-Blunt-E1(cry4Ba-eGFP) reassembled expression vector into E.coli BL21.
08-11 Got recombinants from plate and pick single colony for amplification culture.
08-12 Use ultraviolet rays (UV) to stimulate the GFP by irradiating bacterial suspension. No fluorescence was observed.
08-13 1、Expression of cry4Ba-eGFP After induced by IPTG, we splited E.coli BL21(cry4Ba-eGFP) and loaded the lysate to SDS-PAGE for electrophoresis. According to the right figure, cry4Ba-eGFP did not express.