Team:SJTU-BioX-Shanghai/Notebook/Lablog2

Calendar

9/1/2011

Zhangbo: 8.31 failed

Nachte:Get Rosseta from WY,we contransformed

9/2/2011

Zhangbo: 9.2

Buf    2ul
DNA   16ul(plasmid)
Nde1   1ul
Xho1   1ul
Total    20ul
37℃ 5hours

9/3/2011

ZhangBo: 9.3

Binding the product together
Transfer the plasmid into the DH5a
10ul into 100ul DH5a
30min on the ice
37℃ 2min and 5min on the ice
220rpm 1 hour 
Tet solid medium for 16 hours to 24 hours

9/4/2011

Chunying: Prepared the 4 flask containing 100ml medium to test the four kinds of bacterial(4S,4L,6S,6L luciferase that under the Amp promoter and the rare codon's tRNA is induced by IPTG.

Zhangbo: Failed

9/5/2011

test the 4AGG codon IPTG+Amp promoter system: 4C-co-transformed 4AGG codon IPTG+Amp-promoter system, IPTG+

6C-co-transformed 6AGG codon IPTG+Amp-promoter system, IPTG+

4S-only 4AGG codon IPTG pReporter, IPTG+

6S-only 6AGG codon IPTG pReporter, IPTG+

The IPTG is added into the bacteria when its OD600 reached about 0.3. Take samples every one hour and last for 7 hours.

ZhangBo: 9.5

RFP PCR
PCR KOD buf  5ul
dNTP        5ul
Mg2+        3ul
F/R(WtRNA-1)    2/2ul
Plasmid(WTM-9) 1ul
KOD           1ul
ddH2O       31ul 
Purified the product.
DNA(above) 10ul
plasmid  5ul
PCR KOD buf  3ul
dNTP        3ul
Mg2+       1.8ul
KOD         1ul
ddH2O      6.2ul

Deal with the PCR result above with the DpnI
1ul enzyme(Fast Digest) into 50ul PCR result for 2 hours
Transfer the plasmid into the DH5a
10ul into 100ul DH5a
30min on the ice
37℃ 2min and 5min on the ice
220rpm 1 hour 
Tet solid medium for 16 hours to 24 hours

9/6/2011

Test the effect of concentration of IPTG on the expression of luciferase. Try different concentration of IPTG:

0.1mM,0.2mM,0.3mM,0.4mM,0.5mM,0.6mM,0.7mM,0.8mM,0.9mM,1.0mM

The bacteria used for test: 4AGG+(Amp-luciferase)+(IPTG-tRNA) 4AGG+(Amp-luciferase) without added tRNA

The result indicated that the concentration of IPTG realy has effects on the expression of luciferase as well as the growth of bacteria. But we cannot find out there is no obvious relationship between the concentration and the amount of luciferase. But when the concentration is too high, the growth of bacteria is blocked.

9/7/2011

ZhangBo:

figure1: RFP mutate colony test

9/8/2011

ZhangBo:

Fig1.RFPM PCR test of the plasmid constructed

9/9/2011

Zhangbo: Co-transfer RFPW-pRS1.1 RFPM-pRS1.1

9/12/2011

Try to introduce get wild type luciferase ( under the amp promoter) and the luciferase gene (amp promoter) that have 2 and 8 AGG codons after the initial codon. Still use the point mutation to conduct it. We try different PCR conditions .

1)20 cycles, 60℃ for annealing, 1ul template+2ul primers ( in 50ul system).

2)20 cycles, 60℃ for annealing, 1ul template+3ul primers ( in 50ul system).

3)30 cycles, 55℃ for annealing, 1ul template+1ul primers ( in 50ul system).

Zhangbo: 9.12 Co-transfer Again Choose the right colonies and grow it in the correct LB liquid medium

9/13/2011

We get the wild type luciferase gene (under Amp promoter) by mutation. But the genes that have 2 or 8 AGG codons are not gotten this time. Redo the point mutation PCR to get the luc genes that have 2 or 8 AGG codons.

Zhangbo: 9.13 Choose the right colonies and grow it in the correct LB liquid medium of the Co-transfer Again Grow the first time for more time.

9/14/2011

We failed again to have the luciferase that have 2 or 8 AGG and trancripted after Amp promoter. At the same time, we get the primers to get the luciferase gene that have 2 or 8 AGG codons but under the T7 promoter.

Zhangbo: 9.14 RFPW turn red RFPM no result.

9/16/2011

We find out that the tRNAAsp-TAG and its corresponding aaRS worked as we excepted. The reporter we use is RFP(inserted 6 AGG). Now we want to test if the aaRS can still charge the tRNA when the anticodon of the tRNA is mutated to UAG. So we design the primers to insert a amber codon after the luciferase gene's initial codon.

9/18/2011

We use point mutation to insert one stop codon after the initial codon of luciferase gene. And later in the day we transfer the plasmid into DH5α.

9/19/2011

The bacteria that has the mutated luciferase(having one stop codon after its initial codon)has grown on the plate.We Pick monoclonal colony and extract the plasmid. Then we sent the plasmid to be sequenced.

In the previous experiment we find out that the curves of 4AGG(T7) and 6AGG(T7) mismatch the anticipation and the result cannot explained by the existing knowledge. We doubted that there is something wrong with the constructed pReporter. We sent the two plasmids to sequence.

9/20/2011

The sequencing result showed that we have successfully insert one stop codon after luciferase gene's initial codon. Then we transformed the plasmid into Er2566 along and co-transformed it with the aaRS+tRNA pairs that we constructed(the tRNA's anticodon is mutated to correspond to the stop codon)

9/21/2011

We get the colonies:1.only have the pReporter(one stop codon after luciferase's initial codon) 2.have the pReporter and the pSwitch(the aaRS and tRNA we constructed) We incubated the monoclones into the medium.

The sequencing of 4AGG(T7)and 6AGG(T7) indicated that the 4AGG(T7) plasmid is right but the 6AGG(T7) plasmid is wrong. Today, we began to reconstruct the 6AGG(T7) plasmid.

9/22/2011

Test if the pReporter we constructed can turn up the expression of luciferase that has one stop codon after the initial codon. The method we used to test the luciferase's activities is the same with we used to use. The result indicated that the pSwitch really worked !!!! And the expression of luciferase is greatly increased when there are the aaRS+tRNA.

The new 6AGG(T7) plasmid is transformed to DH5α this morning and we pick the monoclonies late in the day.

9/23/2011

Extract 6AGG(T7) plasmid and send it to be sequenced.

9/24/2011

The result of 6AGG(T7)'s sequencing indicated that the luciferase is inserted 4 AGG codons after the gene's initial codon instead of 6 AGG codons. By examining the previous work, we found out that the primers used for getting the 6AGG(T7)plasmid is wrong!!! It is the same with the primers used for 4AGG(T7). We syntheties the new primers as soon as possible.

9/26/2011

We constructed the 6AGG(T7) plasmid again.

9/27/2011

Reporter for Quantitative Analysis + tRNA Modulator.

2AGG(T7 promoter)+tRNA(lac promoter).

8AGG(T7 promoter)+tRNA(lac promoter).

Every test point have three parallele goups

9/28/2011

We finaly get the right 6AGG(T7) plasmid. And we transform it solely and co-tranRMAsform it with the pSwitch(IPTG+tRNA).

9/29/2011

Reporter for Quantitative Analysis + tRNA Modulator.

4AGG(Amp promoter)+tRNA(lac promoter).

8AGG(Amp promoter)+tRNA(lac promoter).

Every test point have three parallele goups.

9/30/2011

Reporter for Quantitative Analysis + tRNA Modulator.

4AGG(T7 promoter)+tRNA(lac promoter).

6AGG(T7 promoter)+tRNA(lac promoter).

Every test point have three parallele goups

Reporter for Quantitative Analysis + aaRS Modulator

one stop codon after the initial codon in luciferase+aaRS modulator

Every kind of bacteria has three parallel groups