Team:Tsinghua-D/Notebook.html

The Gantt Chart of process of the project

	August
	Sun	Mon	Tue	Wed	Tue	Fri	Sat
				1	2	3	4
	5	6	7	8	9	10	11
	12	13	14	15	16	17	18
	19	20	21	22	23	24	25
	26	27	28	29	30	31

September
Sun	Mon	Tue	Wed	Tue	Fri	Sat
30						1
2	3	4	5	6	7	8
9	10	11	12	13	14	15
16	17	18	19	20	21	22
23	24	25	26	27	28	29

2012.2-2012.4
Conduct literature research.

2012.4-2012.8
Learn Vienna RNA Package.
Learn physical and biological background knowledge.
Write RNAThermo.

2012.8.14

Get ‘1st RNAT + eGFP’ gene from the standard part BBa_I714891 by a three-round overlapping PCR. The result of the first round is negative due to insufficient DNA templates.
Prepare for transformation of the standard part BBa_I714891 to bacteria.

2012.8.15

Transform the BBa_I714891 to the bacteria
Pick single colony of the bacteria harboring BBa_I714891 plasmid.

2012.8.16

Conduct the first round of overlapping PCR of the gene ‘1st RNAT + eGFP’.
Culture the bacteria harboring BBa_I714891 plasmid.

2012.8.17

Extract BBa_I714891 plasmid from bacteria.
Digest the obtained BBa_I714891 plasmid with Pst1 and EcoR1, however the band is unclear.
Take the BBa_I714891 plasmid as template to do the first round overlapping PCR of the gene ‘1st RNAT + eGFP’.
A gradient of PCR annealing temperatures of the gene ‘1st RNAT + eGFP’ are tested to find the optimal one.

2012.8.18

Purify product of the first round overlapping PCR of the gene ‘1st RNAT + eGFP’.
Repeat the PCR and electrophoresis verification procedure.

2012.8.19

Conduct the second round overlapping PCR of the gene ‘1st RNAT + eGFP’.
61℃ is proved to be the best PCR annealing temperature of the gene ‘1st RNAT + eGFP’.

2012.8.20

Conduct the third round overlapping PCR of the gene ‘1st RNAT + eGFP’ and electrophoresis verification procedure.

2012.8.21-8.22

Purify the product of the third round overlapping PCR of the gene ‘1st RNAT + eGFP’ by gel extraction.

2012.8.23

Digest the plasmid and the PCR product of the gene ‘1st RNAT + eGFP’ with EcoR1 and Pst1.
Ligate the two digested DNA.

2012.8.24

Test shows the construction of the plasmid fail.

Retry the ligation

2012.8.25

Ligation and transformation of ‘the 1st RNAT + eGFP’ are done.
Replicate pSB1C3 plasmid with the protocol given by iGEM.
Adapt pET-Duet plasmid as expression vector instead of pSB1C3.
Prepare for the lysozyme experiment.
Transformation of the pSB1C3 plasmid is done.

2012.8.26

Pick the single and positive colony of ‘1st RNAT + eGFP’.

2012.8.27

Get ‘signal peptide + lysozyme’ gene by a three-round overlapping PCR.
Conduct 1st and 2nd round of the overlapping PCR of the gene ‘1st RNAT + signal peptide + lysozyme’.

2012.8.28
Amplify the PCR product of the gene ‘1st RNAT + signal peptide + lysozyme’.

2012.8.29

Prepare for the in-line probing.

2012.8.30

Conduct the in-line probing.

Prepare for the ‘2nd RNAT + eGFP’ and ‘3rd RNAT + eGFP’ experiments.

2012.8.31

Learn fluorescence signal detection and RNA in vitro transcription procedure in the in-line probing method.
Conduct the ‘2nd RNAT + eGFP’ and ‘3rd RNAT + eGFP’ experiments.

2012.9.1

Send the plasmid harboring ‘1st RNAT + eGFP’ gene for sequencing
The RNA in vitro transcription is done.
Conduct the ‘2nd RNAT + eGFP’ and ‘3rd RNAT + eGFP’ experiments.
Conduct the ‘1st RNAT + signal peptide + lysozyme’ experiment.

2012.9.2

Send the plasmid harboring ‘2nd RNAT + eGFP’ gene and ‘3rd RNAT + eGFP’ gene for sequencing.
Conduct the ‘1st RNAT + signal peptide + lysozyme’ experiment.

2012.9.3

Overlapping PCR of the ‘1st RNAT + signal peptide + lysozyme’ gene is done.

Obtain ‘1st RNAT + eGFP’ result, however not convincing.

2012.9.5

Obtain ‘2nd RNAT + eGFP’ result, convincing result.
Obtain ‘3rd RNAT + eGFP’ result, convincing result.

2012.9.8-9.22

Repeat ‘1st RNAT + eGFP’, ‘2nd RNAT + eGFP’ and ‘3rd RNAT + eGFP’ results.
Conduct the in-line probing.
Standard parts are made and sequenced.

2012.9.23-9.26

Process the data, write for the report.
Prepare for the ‘2nd RNAT + signal peptide + lysozyme’ and ‘3rd RNAT + signal peptide + lysozyme’ experiments.