Team:Heidelberg/Templates/Indigoidine week15

From 2013.igem.org

Contents

CPEC Conditions Assay (Ralf)

We performed CPEC assembly of pRB13 with eight different CPEC conditions to see whether we get difeerent results.

Table 11.1 CPEC mix for assembly of pRB13

Annealing Temp [°C]Cycles
51015
532: 10 ul; 4 ul backbone; 1 ul CCDB
553: 10 ul; 4 ul backbone; 1 ul CCDB
7: 20 ul; 8.5 ul backbone; 1.5 ul CCDB
4: 10 ul; 4 ul backbone; 1 ul CCDB
6: 20 ul; 8.5 ul backbone; 1.5 ul CCDB
8: 10 ul; 4.5 ul backbone; 0.5 ul CCDB
5: 10 ul; 4 ul backbone; 1 ul CCDB
571: 10 ul; 4 ul backbone; 1 ul CCDB

Table 11.2 CPEC Assembly of pRB13

BioRad T100
cyclestemp [°C]time [s]
19810
5/10/15981
53/55/575
72120
172300
112-

100 ul of competent TOP10 were transformed with 5 ul of reaction product and incubated for 30 hours at 37 °C.


10 ul; 4 ul backbone; 1 ul CCDB 10 ul; 4 ul backbone; 1 ul CCDB 10 ul; 4 ul backbone; 1 ul CCDB 10 ul; 4 ul backbone; 1 ul CCDB 10 ul; 4 ul backbone; 1 ul CCDB 20 ul; 8.5 ul backbone; 1.5 ul CCDB 20 ul; 8.5 ul backbone; 1.5 ul CCDB 10 ul; 4.5 ul backbone; 0.5 ul CCDB


Apparently there is no big difference using various CPEC conditions. We will continue with the standard protocol.

PPTase test (Ralf)

pRB3-10 are plasmids containing indC (pRB-3-6) or bpsA(pMM64) (pRB7-10) and a PPTase (sfp, svp, svp(pMM65), entD).

This week the blue colonies are screened and sequenced for validation.

colony PCR screening #1

Two blue colonies are picked from each plate and screened with the primers of the PPTase they should have and the

ones which they could have if something went wrong. PPTase PCR products werde used as a positive control and TOP10

without any plasmid as a negative control. So numbers 5 to 18 are subdivided into alpha and beta.

PPTase to be screened
templatesfp (RB35/36)svp (RB29/30)svp(pMM65) (RB25/26)entD (RB33/34)
1
2
3
4
5
6
7
13 14 15 8
9
10
11
1617 18 12
1920 21 22
BioRad T100
cyclestemp [°C]time [s]
1120
309560
6530
7260
172300
112-
Loading Scheme
5a5b9a9b1192-log6a6b10a10b220
7a7b11a11b3212-log8a8b12a12b422

The gel is not conclusive. Therefore I will repeat the screening but screen every colony for all PPTases.

MiniPrep of pRB4-10

5 ml liquid cultures of TOP10+pRB4-10 have been miniprepped using QIAquick miniprep kit. Only blue cultures have

been prepped (except pRB9 because there was no blue culture). pRB4-6 and pRB10 were prepped in duplicates (liquid

cultures of two different clones) whereas there are only single preps of pRB7-9. This is because pRB3-6 contain

indc ahich we will use for further studies. pRB6 and 10 contain entD, which is the E. coli endogenous PPTase and

was previously declared to be "inefficient" in activating bpsA [Takahashi 2007].

Concentrations of the prepped plasmids have been measured using ??ThermoScientific NanoDrop

plasmidversionconcentration [ng/ ul]
pRB4alpha289.7
beta278.7
pRB5alpha228.3
beta348.9
pRB6alpha428.0
beta432.2
pRB7alpha187.3
pRB8alpha768.2
pRB9alpha142.5
pRB10alpha112.0
beta212.0
water after measurement0.7

Minipreps were reduced to a concentration of about 50 ng/ ul for sequencing and 1 ng/ ul for further studies.

MiniPrep PCR screening #2

PPTase to be screened
templatesfp (RB35/36)svp (RB29/30)svp(pMM65) (RB25/26)entD (RB33/34)
TOP10+pRB45 67 8
TOP10+pRB5 9 1011 12
TOP10+pRB613 14 15 16
TOP10+pRB7 17 18 19 20
TOP10+pRB821 22 23 24
TOP10+pRB9 25 2627 28
TOP10+pRB10 2930 31 32
sfp 33
svp 34
svp(pMM65) 35
entD 36
TOP10 3738 39 40


BioRad T100
cyclestemp [°C]time [s]
195120
309560
6030
7260
172300
112-
Loading Scheme
5a5b9a 9b13a13b17212529a29bsfp2-log6a6b10a10b14a14b18222630a30bsvp
7a7b11a11b15a15b19232731a31bsvp(pMM65)2-log8a8b12a12b16a16b20242832a32bentD

pRB9 is negative. All screens are positive in the svp(pMM65) and entD section. In entD this could be due to genomic

DNA in the miniprep. svp(pMM65) is also positive in all plasmids, which is strange. Maybe the PCR conditions have

been too tolerant. Nevertheless the bands of the plasmids which should carry the respective PPTase appear to be

stronger. We will sequence the plasmids anyways.

Sequencing of pRB3-10

plasmid minipreps have been sequenced by GATC [reference] using the sequencing primers VF2 and VR. The sequencing

results show that all the inserts are correct except for pRB9, which isn't a pity because we have previously shown

with pKH1-2 that the combination of bpsA(pMM64) and svp(pMM65) successful produces indigoidine.

plasmidPrimerind synthetase/ PPTasesequencing result/ reference
pRB3VF2indC
seq2
VRsfp-rev
seq2
pRB4VF2indC
seq2
VRsvp(pMM65)-rev
seq2
pRB5VF2indC
seq2
VRsvp-rev
seq2
pRB6VF2indC
seq2
VRentD-rev
seq2
pRB7VF2bpsA
seq2
VRsfp-rev
seq2
pRB8VF2bpsA
seq2
VRsvp(pMM65)-rev
seq2
pRB9VF2bpsA
seq2
VRsvp-rev
seq2
pRB10VF2bpsA
seq2
VRentD-rev
seq2


In the following pRB4-beta, pRB5-alpha, pRB5-beta, pRB6-alpha and pRB10-alpha have been selected so that there are

single versions of pRB3-10 for furhter experiments.

Glycerols stocks pRB3-10

3 stocks each were made using 2 mL liquid cultures (48 h at 37 °C 200 rpm) of TOP10+pRB3-10, respectively.