Revision as of 18:22, 4 September 2008

Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter

Analysis of the transformation we did yesterday

Number of colonies and flurorescence

Ligation name	Description	Antibio	Number Colonies observed	Fluorescence	Comments
Ligations
L150	D105(BV) - D146(BI) pLas - rbs-TetR-GFP Tripart	Amp	3	No	Problem
L151	D147(FI) - D125(FV) rbs-LasR - Double terminator	Kan	21	No	OK
Controls
C1	D105(BV)	Amp	150	NO	OK
C2	D125(FV)	Kan	2	No	OK
Positive Control	pUC19	Amp	416 (efficiency 4.10^7)	No	OK

PCR Screening

Protocol

L150 and L151

Well	Sample	Expected size	Measured size
1	1kb ladder
2	Negative control (pUC19)	nothing	nothing
3	Positive control (MP101.1)	768 pb	800pb
4	L150.1	1992pb	400pb
5	L150.2
6	L150.3
7	L151.1	1229pb	1200pb
8	L151.2
9	L151.3
10	L151.4
11	L151.5
12	L151.6
13	L151.7
14	L151.8
15	100pb ladder

Conclusion : 
L150 doesn't success, measured size match with the promoter size only, 
 we will check the size of L101 by screening and if it doesn't match with the good size (1827pb) 
 we will try again the ligation : rbs-TetR (S03879) with GFP tripart (E0840).
L151 is ok we have to put a strong promoter before the part rbs-lasR-double terminator :GREAT !

Minipreps & Stocks

Cultured in 7,5ml of LB with a thoothpick of a colony.
Culture O/N at 37°C of :

Miniprep Name	Ligation name	Antibio	Biobricks	Description
MP161.1	L150.1	Amp		pLas - rbs-TetR-GFP tripart
MP161.2	L150.2
MP161.3	L150.3
MP162.1	L151.1	Kan		rbs-LasR - Double terminator
MP162.2	L151.2
MP162.3	L151.3

Analysis of the other transformation we did yesterday

Evaluation of the number of colonies

Ligation name	Number of colonies	Autoligation control
L 143 (Kan)	0	0
L 144 (Kan)	0	0
L 145 (Amp)	452	430
L 146 (Amp)	~ 2000	430
L 147 (Amp)	~3500	>5000

Remarks: 

    L143 and L144 did not work once again. Maybe there is a problem with the digestion,  
 because the primers  used present only two nucleotides after the restriction sites. what we should  
do is cutting pfliL with xbaI and SpeI. As the vector will autoligate, we should use a phosphatase  
 to prevent this phenomenon. 

    For L147 and its control (T4), we do not observe any red fluorescence. It means that the digestion work well, because if it was not digested, the strong promoter J23100  
should  express mRFP!  
 We will repeat all the ligation anyway.

**Construction of OmpR+RBS and EnvZ+RBS: transformation**

Evaluation of the number of colonies

Ligation name	Number of colonies	Autoligation control
L 148 (Amp)	~10000	~2000
L 149 (Amp)	~750	~2000

@@ Line 188: / Line 188: @@
   Remarks: <br>
       L143 and L144 did not work once again. Maybe there is a problem with the digestion,  <br> because the primers  used present only two nucleotides after the restriction sites. what we should  <br>do is cutting pfliL with xbaI and SpeI. As the vector will autoligate, we should use a phosphatase  <br> to prevent this phenomenon. <br>
-      For L147 and its control (T4), we do not observe any red fluorescence. <br>
+      For L147 and its control (T4), we do not observe any red fluorescence. It means that the digestion work well, because if it was not digested, the strong promoter J23100  <br>should  express mRFP!  <br> '''We will repeat all the ligation anyway'''.
-     It means that the digestion work well, because if it was not digested, the strong promoter J23100  <br>should  express mRFP!  <br> We will do the screening anyway.
 =='''Construction of OmpR*+RBS and EnvZ*+RBS: transformation'''==

Team:Paris/August 17

From 2008.igem.org

Revision as of 18:22, 4 September 2008

Contents

Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter

Analysis of the transformation we did yesterday

Number of colonies and flurorescence

PCR Screening

Minipreps & Stocks

Analysis of the other transformation we did yesterday

Evaluation of the number of colonies

**Construction of OmpR+RBS and EnvZ+RBS: transformation**

Evaluation of the number of colonies