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From 2012.igem.org

Week 9 bogged, Subversion and start again

2012/8/13 Monday, August 13, 2012

Yu Jianing and Yan came to Prof. Chen Xin for MD (molecular dynamics simulation, suggested by PKU iGem) modeling in RNA scaffold project. Prof. CHEN pointed out that it's so hard and costing for us green hand undergraduates to manipulate MD, which could be replaced by RNA prediction software and ODE modeling with matlab.

Zhang and Chen transform a group of D0, FA and FB with different combination.

LIU Xiao comes to carry out point mutation experiment to modify origin D0 in PCR method. He intended to build a library. However, we have only one month left, too short to finish the library.

Yan's fluorescent test with part K537009 is modestly successful. Most excitingly, Yan and Chen capture a love heart (or you can refer to it as a smiling smile) imaging by E.coli.

2012/8/14, Tuesday 14, 2012

Chen makes competent cell with BL21 star DE3 borrow from Zhejiang Sci-Tech University.

2012/8/15 Wednesday, August 15, 2012

Prof. SHAO Jianzhong is willing to let us use the Synergy hybrid reader in his lab.

Chen and Zhang test fluorescence of D0, FA,FB,FA+D0,FA+FB,FA+FB+D0 with IPTG concentration gradient but find out that the fluorescence intensity are nearly no difference comparing with scaffold and without scaffold surprisingly.

2012/8/16 Thursday, August 16, 2012

After senior fellow's advises, however, we still can't see the result in < Organization of Intracellular Reactions with Rationally Designed RNA assemblies>.

Yan tried the parts of Theophylline riboswitch with YFP Venus reporter and it worked. (Though excite 515nm emit 528 mn is too close for Synergy hybrid reader to detect and we change it into 505nm and 535nm.)

You can see the fluorescence intensity increasing with Theophylline concentration.

All night talks and strange Sequencing results make us skeptical about the FAFB plasmids sent by French lab (which is crucial to our project). Maybe there's something wrong with our raw material.

2012/8/17 Friday, August 17, 2012

Today we faced mega-disaster that most of us thought the plasmids: FA & FB sent early August are not what we were asking for. Dr. Delebecque is so helpful and patient but he's away from his French lab now. He sent us an email providing a lot of information we need if we construct the plasmid on our own. So we have to go back and start from the very beginning. Chen, Zhang and Liu try to redesign the primers; Prof. Shao lent us the eGFP. Our advisor, Li Xin comes to discuss with us the technology of construct.

"You must be ready to burn yourself in your own flame: how could you become new, if you had not first become ashes?" --When Nietzsche Wept

2012/8/18 Saturday, August 18, 2012

1 We bought commercial plasmids pCOLADuet?-1 Vector and pCDFDuet-1 Vector, and streak plate them respectively in Spectinomycin, Kanamycin and LB plate.

2 The part "2012 plate 1 15P" can't be transformed, as Zhang and Yan tried it twice.

2012/8/19 Sunday, August 19, 2012

1 plasmid miniprep:

Plasmid Concentration A260/280 A260/230

pCOLADuet?-1(1) 29.9 ng/μL 1.96 1.21

pCOLADuet?-1(2) 25.6 ng/μL 2.07 1.19

pCOLADuet?-1(3) 28.0 ng/μL 2.07 1.15

2 LIU Xiao continues his point mutation experiment to modify origin D0 in PCR and build a library of changed RNA scaffold with different aptamer arm length.

He makes a new RNA scaffold, Nucleic Acid Electrophoresis as follows.

First step:

Second step:

First step product is due in 50bp and second is due in 200bp. But the Electrophoresis effect is not good.

3. Freshmen come to us and we taught them some methods for using Library Resources and making it in college.