Team:FAFU-CHINA/Notebook

Week 1 (5.16-5.22）

Before thefirst week, we cultivated Chlamydomonasreinhardtii.

We sent our reserved parts to SYSU-CHINAteam and ShanghaiTechChina_B team to help them to finish project.

BBa-k325903 Plate1 2L

BBa-k 415023 Plate1 2P

BBa-k325909 Plate1 4L

BBa-k934012 Plate1 5H

BBa-k934022 Plate1 5J

BBa-k934026 Plate1 5L

BBa-k516011 Plate1 9G

BBa-k546001 Plate1 12F

BBa-k584002 Plate1 13E

BBa-k594001 Plate1 18I

BBa-k381001 Plate1 4B

BBa- E0040 Plate4 13L

Wedistributed questionnaires about mosquito-borne diseases in China.

Week 2（5.23-5.29）

We designed primers for Cry4a,Cry10a, Cry11a,Cyt1,Cyt2,EGFP genes.The genes from Bacillus thuringiensis serovar israelensis strain BRC-LLP29

Name Sequence length

Cry11Aa4-In-BglII-Short-F CCATACAGTTCTAGAGAATGAATTATATGGAAGATAGT 38

Cry11Aa4-In-NdeI-Short-R CGGAGCGGAATCATATCGGATTAAT 25

Cry4Aa4-In-BglII-Short-F CCATACAGTTCTAGAGAATGAATCCTTATCAAAATAAA 38

Cry4Aa4-In-NdeI-Short-R CGGAGCGGAATCATATTCACTCG 23

Cry10Aa4-In-BglII-Short-F CCATACAGTTCTAGAGAATGAATCCATATCAAAATAAG 38

Cry10Aa4-In-NdeI-Short-R CGGAGCGGAATCATATATACAGATTGA 27

Cry4b-In-BglII-Short-F CCATACAGTTCTAGAGAATGTGTGAAAATAACCAA 35

Cry4b-In-NdeI-Short-R CGGAGCGGAATCATATTTATTTTGA 25

Cyt1-In-BglII-Short-F CCATACAGTTCTAGAGAATGGAAAATTTAAATCATTGT 38

Cyt1-In-NdeI-Short-R CGGAGCGGAATCATATTTAGAGGGT 25

Cyt2-In-BglII-Short-F CCATACAGTTCTAGAGAATGCACCTTAATAATTTGAAT 38

Cyt2-In-NdeI-Short-R CGGAGCGGAATCATATTTATTTTATTGG 28

EGFP-In-BglII-F CCATACAGTTCTAGAGAATGGTGAGCAAG 29

EGFP-In-NdeI-R CGGAGCGGAATCATATCTTGTACAG 25

Week 3（5.30-6.5）

Molecular cloning of Cry4a,Cry4b,Cry10a,Cry11a,Cyt1,Cyt2,EGFP genes.Vector is pChlamy-3.

PCR

goto cycles

1 Pre-denaturation 94°C 2min

2 Denaturation 98°C 10sec

3 Extension 68°C 30 sec/kb 2 25~45

4 Final Extension 68°C 5min

template Product length Result

Cry11A Bt-1 1959bp X

Cry4Aa Bt-1 3573bp X

Cry4b Bt-1 1818bp X

Cry10A Bt-1 2058bp X

Cyt1 Bt-1 780bp X

Cyt2 Bt-1 819bp X

EGFP pK7GWF2 747bp √

Analysis of Negative Result： Annealingreaction was not set.

Week 4(6.6-6.11)

PCR

goto cycles

1 Pre-denaturation 94°C 2min

2 Denaturation 98°C 10sec

3 Annealing 58℃ 30sec

4 Extension 68°C 30 sec/kb 2 25~45

5 Final Extension 68°C 5min

Product length Result

M Trans2KPlus DNA Ladder

1 Cry11A Bt-2 1959bp √

2 Cry4Aa Bt-2 3573bp

3 Cry10A Bt-2 2058bp √

4 Cry4b Bt-2 1818bp √

5 Cyt1 Bt-2 780bp √

6 Cyt2 Bt-2 819bp √

7 Cry11A Bt-3 1959bp √（Non-specific）

8 Cry4Aa Bt-3 3573bp

10 Cry10A Bt-3 2058bp √（Non-specific）

9 Cry4b Bt-3 1818bp

11 Cyt1 Bt-3 780bp √（Non-specific）

12 Cyt2 Bt-3 819bp √

13 Cry11A Bt-4 1959bp X（Non-specific）

14 Cry4Aa Bt-4 3573bp （Non-specific）

16 Cry10A Bt-4 2058bp √（Non-specific）

15 Cry4b Bt-4 1818bp √（Non-specific）

17 Cyt1 Bt-4 780bp √（Non-specific）

18 Cyt2 Bt-4 819bp √（Non-specific）

M Trans2KPlus DNA Ladder

1 Cry11A Colony 1959bp X

2 Cry4Aa Colony 3573bp X

3 Cry4b Colony 1818bp √

4 Cry10A Colony 2058bp X

5 Cyt1 Colony 780bp X

6 Cyt2 Colony 819bp √

7 Cry11A Colony 1959bp X

8 Cry4Aa Colony 3573bp X

9 Cry4b Colony 1818bp X

10 Cry10A Colony 2058bp X

11 Cyt1 Colony 819bp √

12 Cyt2 Colony 1959bp √

M Trans2KPlus DNA Ladder

PCR

goto cycles

1 Pre-denaturation 94°C 2min

2 Denaturation 98°C 10sec

3 Annealing 58℃ 30sec

4 Extension 68°C 30 sec/kb 2 25~45

5 Final Extension 68°C 5min

template Primer-F Primer-R Product length Result

1 EGFP pK7GWF2 EGFP-In-BglII-F EGFP-In-NdeI-R 747bp √

2 EGFP pK7GWF2 EGFP-In-BglII-F EGFP-In-NdeI-R 747bp √

Gel Extraction

Result

1 EGFP √

2 EGFP √

estriction enzyme digestion

enzyme template

1 BglII&NdeI pChlamy-3

2 BglII&NdeI pChlamy-3

Purificationof enzyme digest product

In-Fusion

10μl TotalVolume

2 μl 5XIn-Fusion HD Enzyme Premix

3μl LinearizedVector

4μl PurifiedPCR Fragment

1μl dH2O(as needed)

Incubate the reaction for 15 min at 50°C, then place on ice.

Continue to the TransformationProcedure.

1 EGFP+pChlamy_3

2 EGFP+pChlamy_3

Week 5(6.12-6.18)

On 17th June, Junhao visited theShenzhen University and their team (SZU-China) after Synthesis Biology Meeting.

PCR：Cry4a,Cry10a, Cry11a,Cyt1,Cyt2

Agarosegel electrophoresis

Purificationof PCR product or Gel extract

Gelextraction

Doubleenzyme digest of plasmid

Purificationof enzyme digest product

Agarosegel electrophoresis

In-fusion

Transformation

Week 6(6.19-6.25)

We try to UV irradiation of Bacillusthuringiensis, We need to determine the Bacillus thuringiensis concentration.

Production of summer anti-mosquitohandbook

Week 7 (6.26-7.2)

UV irradiation of Bacillus thuringiensis.

Week 8 (7.3-7.9)

UV irradiation of Bacillus thuringiensis.

We completed the first edition of thehandbook.

Week 9(7.10-7.16)

Codon optimization of Chlamydomonas reinhardtii by synbio-tech

Week 10(7.17-7.23)

Gene synthesis by synbio-tech

Week 11(7-24.7.30)

Cloning of the genes with codonoptimization in Chlamydomonas reinhardtii ，Vector is pChlamy-3.

Week 12(7.31-8.6)

We got a chance to study at departmentof Health Education in Jiangxi Provincial Institute of Parasitic Disease forhalf a day.

Week 13(8.7-8.13)

We noticed that the fluorescence valuein treatment group was higher than the positive control group about JNFLS-CHINAhigh school team’s project. We had a talk about it by social media. To explorethe potential cause, we advised them to use Flow Cytometer (FCM) to gatherdata. And we helped them design the protocol of experiment with details. If youare interested in the details, you can visit this link: <a href="https://2016.igem.org/Team:JNFLS_China/experiments">https://2016.igem.org/Team:JNFLS_China/experiments</a> and results

Week 14(8.14-8.20)

During the G20 Summit held in Hangzhou,we shared our labs with them in summer.

Week 15(8.21-8.27)

We Identified the production of theclone.

Transforming Chlamydomonas reinhardtii by Electroporation.

Week 16(8.28-9.3)

We used confocal microscopy to observethe expression of GFP, and this transformation was negative.

We participated in the CCIC (CentralChina iGEM Consortium) held in Zhongshan University in Guangzhou.

Week 17(9.4-9.10)

Transforming Chlamydomonas reinhardtii by Electroporation again.

Week 18(9.11-9.17)

We helped NEU-China to test theexpression of tCas9-CIBN and got the accurate data about the inducedconcentration of arabinose.

Week 20(9.18-9.24)

We screened the transformants of C. reinhardtii. The greens in the figureare positive for transformation.

We designed the qPCR primers use ofdetection Cry4a,Cry10a,Cry11a,Cyt1,Cyt2 genes expression.

cry4a-qPCR1-F-133 ACGACCAGATGGAGGCCAAG 20

cry4a-qPCR1-R-133 TACTGGATCTGGGCCAGGGT 20

cry4a-qPCR2-F-70 CCAGGACAGCCACCAGTTCA 20

cry4a-qPCR2-R-70 CACGCCGATGTTCTCGTTGG 20

cry10a-qPCR1-F-94 CGCAACAAGCCCATCGACAA 20

cry10a-qPCR1-R-94 CCTCGCTGCTGTTGCTGAAG 20

cry10a-qPCR2-F-84 TTCGGCTACGTGACCTTCCC 20

cry10a-qPCR2-R-84 GGTGCCGTACAGGGTCATCA 20

cry11a-qPCR1-F-85 CGAGTGGGTGGACTTCGTGA 20

cry11a-qPCR1-R-85 GATGCTGAACAGGGCGTTGG 20

cry11a-qPCR2-F-69 CCAACGCCCTGTTCAGCATC 20

cry11a-qPCR2-R-69 ACAGCTGGCTCAGGTACCAC 20

cyt1-qPCR1-F-136 ACAACGTGCTGTTCGCCATC 20

cyt1-qPCR1-R-136 TTGTAGCTGGCGGAGTCCTG 20

cyt1-qPCR2-F-123 AACAAGGTGCTGGAGGTGCT 20

cyt1-qPCR2-R-123 GGCCTCGTTCTTCTGGGTGT 20

cyt2-qPCR1-F-137 CAGACCATCGAGGTGAGCGT 20

cyt2-qPCR1-R-137 GGCTCCAGGTTGGTGAAGGT 20

cyt2-qPCR2-F-99 GCCTTCGAGATCACCGTGGA 20

cyt2-qPCR2-R-99 CACGGTCAGGGCCTTCATCT 20

egfp-qPCR1-F-64 GGCCACAAGTTCAGCGTGAG 20

egfp-qPCR1-R-64 TCAGGGTCAGCTTGCCGTAG 20

egfp-qPCR2-F-140 GCCGACAAGCAGAAGAACGG 20

egfp-qPCR2-R-140 TAGTGGTTGTCGGGCAGCAG 20

Week 21(9.25-10.1)

Week 22(10.2-10.8)

ExtractRNA

RT-PCR

QPCR

Week 23(10.9-10.15)