UPDATE 09/18/2013

Molecular construction of the up stream elements(regulating parts,protein tTA，rtTA，tTA advanced ，and eGFP for contrast ) of tet systems.

Introduction

Team members in Charge：Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)

Author: Dawei He

Team members in Charge：Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)

Despite a good comprehension of the knowledge of Molecular biology and its relating experimental technologies，We never came to and stay in a Molecular biology lab and were completely lack of any experience. To achieve the designated tasks in extremely insufficient time(less than 3 months),We had to learn fast and start our work independently as soon as possible.

2013.5.20-5.23

Contents: Contact and learning of Molecular construction

Team members in Charge：Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)

1. DNA extraction techniques: Plasmid extraction, DNA clean up(extraction from PCR or restriction endonuclease digestion systems,extraction from Ag gel, etc)

2. PCR techniques:

Primer(for molecular construction or sequencing) designing, PCR for molecular construction, colony PCR for verification.

3. Endonuclease digestion

2013.5.24 to 5.31

Contents: First plasmid construction：pCDNA3.0-PGK-BSD

Annotation：pCDNA3.0 is a backbone plasmid ideal for transient expression in our designated cell lines including HEK293T,HepG2 and Bocs. All elements of our project are first to be incised into pCDNA3.0 and tested during transient expression in cell lines. Driven by promoter PGK,BSD express (!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1)

Figure 1.Backbone plasmid pCDNA3.0(completed featured)

Figure 2.pCDNA3.0-PGK-BSD

construction details:

1.PCR of PGK-BSD(915bp in length) from donor plasmid(as PCR template)

Primers：Forward primer with restriction site BamHI and necessary base pairs from 5’ end

Reverse primer with restriction site XhoI and necessary base pairs from 5’ end

Bases pairs should appear at both flanks of restriction sites to provide anchoring sites for endonuclease,3’ end of the restriction sites in a primer is already covered by base pairs, so some base pairs must be added to its 5’ end. Without such manually added base pairs from 5’ end, efficiency of endonuclease digestion of PCR product will be significantly reduced.

1.1 PCR system set up

Table 1 Oncogenic risks associated with methods of inducing pluripotency in somatic cells

5×fastpfu PCR buffer	5μl
2.5mM dNTP：	2.5μl
10uM PB F primer	0.8μl
10uM PB R primer	0.8μl
Template(plasmid)	4μl(5.36ng/ul,21.4ng in total. Acceptable quantity from 5-30ng)
Fastpfu DNA polymerase	0.5μl
ddH20	11.4μl(up to 25ul)

1.2 temperature program set up

Annealing temperature in main PCR cycles =Primer Tm-5℃

Pre-cycles with lower annealing temperature in PCR process of primers with restriction sites(or any primers that include bases pairs not complementary to their original templates ) are necessary to achieve both specificity and quantity demand of PCR product.

During the first PCR cycle, primer binds only and incompletely to its original templates, so tm is lower compared to the one when primer binds total- complementarily to its product generated since the first cycle, which was why the idea of adding pre-cycles before main cycles came up. Although annealing temperature in pre-cycles can be calculated to details, the temperature 15℃ below annealing temperature in main cycles will be quite universal.

Primer binding to its original template

Primer binding to its PCR product

1.3 Endonuclease digestion of backbone plasmid and insert fragment（PCR product）

PCR product PGK-BSD was purified using PCR clean up kit.

10xFermentas Buffer G	3μL
pCDNA3.0	2μL（3500ng）
BamHI(Fer) （100% activity）	1μL
XhoI（Fer）（50-100% activity）	2μL
ddH20	22μL（up to 30μL）

10xFermentas Buffer G	3μL
PGK-BSD	9μL（900ng）
BamHI(Fer) （100% activity）	1μL
XhoI（Fer）（50-100% activity）	2μL
ddH20	15μL（up to 30μL）

Both digestion systems were incubated in 37℃ for 2h

Figure 3.

Result：backbone plasmid pCDNA3.0 was fully digested，but as the fragment excised from pCDNA3.0 was too short(which means too small in quality because it has the same amount of substance as the backbone’s) in length(less than 50bps),it cannot be identified in agarose gel-electrophoresis. When digested in a least one restriction site, plasmid turns from supercoil status to linearized and has lower mobility in gel-electrophoresis, so it can only be judged that backbone was definitely complete digested in at least one restriction site. Such assessment applies also to digestion of PCR product from whom the excised parts are almost never more than 50bps.

1.4 Ligation

Ligation type：1 vector+1 insert fragment

Desired ratio of the amount of substance：

Vector：insert fragment =1:5

10xFermentas T4 ligation buffer	2μL
BamHI-(pCDNA3.0)-XhoI	1μL（60ng）
BamHI-（PGK-BSD）-XhoI	2μL（60ng）
Fermentas T4 ligase	1μL
ddH20	12μLup to 20μL）

Ligation for 2h at room temperature(25℃)

1.5 Transformation of ligation product

Transformation system

DNA（ligation system）	2μL
Competent E.coli（Top10 strain）	50μL

After 2h ligation，ligation system,as well as Competent E.coli taken from -80℃ storage， was first incubated in ice for 5min before mixing together.

Transformation system continued to be incubated in ice for 30mins,42℃heat shock for 60s，then again ice incubation for 5mins.

Transformation system was added 600μL antibiotic-free LB medium and put in 37℃ thermostat shaker for 30mins-resuscitation（expression of Amp resistance protein）

Transformation system was then centrifuged at 130,000g for 3mins.All but little supernatant was kept to suspend the E.coli，which was later transferred to the LB agar plate. The plate was put in 37℃ thermostat incubator overnight for colonies growth.

1.6 Verification of the colonies（each as a single clone）

Figure 4.

colony PCR set up

Table 1 Oncogenic risks associated with methods of inducing pluripotency in somatic cells

5×fastpfu PCR buffer	5μl
2.5mM dNTP	2.5μl
10uM PGK-BSD F primer F primer	0.8μl
10uM PGK-BSD R primer	0.8μl
Template （single-clone colony）	1μL（taken from the single-clone Colony suspended with 5μL ddH20
Fastpfu DNA polymerase	0.5μl
ddH20	11.4μl(up to 25ul)

Figure 5

Result：Clear band around 1000bp appeared for every 12 clone.

1.7 Plasmid extraction and verification

6 of the single-clone colonies were selected and added to 5ml Amp-LB medium and put to 37℃ thermostat shaker.

The bacteria were centrifuge after 16h-growth in the thermostat shaker.Plasmids were extracted using endotoxin-free kit.

The plasmids were later digested by BamHI and XhoI for verification.

10xFermentas Buffer G	2μL
pCDNA3.0	0.5μL（400ng）
BamHI(Fer) （100% activity）	0.3μL
XhoI（Fer）（50-100% activity）	0.6μL
ddH20	16.6μL（up to 30μL）

Figure 6

Result: Clear band around 1000bp appeared. Plasmids from every 4 clones were verified.

Plasmids were later sent for sequencing

2013.6.1-6.14

Construction of pCDNA3.0-pEF1a-tTA-PGK-BSD

pCDNA3.0-pEF1a-rtTA-PGK-BSD

pCDNA3.0-pEF1a-tTA advanced-PGK-BSD

pCDNA3.0-pEF1a-eGFP-PGK-BSD

Figure.7

PCR system set up

5×fastpfu PCR buffer	5μl
2.5mM dNTP：	2.5μl
10uM PB F primer	1.6μl
10uM PB R primer	1.6μl
Template(plasmid)	2ul(50ng)
Fastpfu DNA polymerase	1.0μl
ddH20	28.8μl(up to 25ul)

Temperature program set up

（1×）	95℃	5min	initial denaturation
（5×）	95℃	20s	denaturation
	1.6μl 52℃	20s	annealing
	72℃ < /td>	35s	elongation
（25×）	95℃	20s	denaturation
	63℃	20s e	annealing
	72℃	35s	elongation
72℃	（1×）	5min
（1×）	4℃	10min

Figure.8

Digestions

Chart1 Chart 2

Incubated in 37℃ for 90mins

Ligation

Ligation type：1 vector+2 insert fragments

Desired ratio of the amount of substance：

Vector：insert fragment 1：insert fragment 2=1：3：3

Verification of single-clone colonies

PCR verification of Result All chosen colonies are positive, so were the sequencing results.

2013.6.18-7.1

construction of pCDNA3.0-pCMV-RIP1

pCDNA3.0-pCMV-RIP1-1X miR122 complete target

pCDNA3.0-pCMV-RIP1-2X miR122 complete target

2013.7.5-7.31

construction of p199-pEF1a-tTA-PGK-BSD

p199-pEF1a-rtTA-PGK-BSD

p199-pEF1a-tTA advanced-PGK-BSD

p199-pEF1a-RIP1-2XmiR122 targets

p199-pEF1a-RIP3-2XmiR122 targets

p199-pEF1a-eGFP-PGK-BSD

Figure.9

A major problem: DNA Recombination of pEF1a 2013.8.1-8.22

Promoter EF1a we used to drive regulationg parts of tet-on or tet-off system are 537bp in length(supported by professor Lei xiao, Zhejiang University).By NCBI blast search，first 349bp of the 537bp-pEF1a was found to be CPPT，one of the essential elements of inserting backbone plasmid of lentivirus. While the function of CPPT cassette inside pEF1a is yet to figure out, the result of molecular construction suggested that CPPT, which exists both inside 537bp-pEF1a and p199 backbone plasmid, has high ratio of recombination that can interfere the normal inserting process of lentivirus vector and the expression of downstream genes.

Figure 10. The recombination zone

High-ratio recombination between the 2 copies CPPT in plasmid p199-pEF1a(537bp)-gene was first suspected as the forward sequencing primer often present 3 kinds of sequencing results: 1.No signal(most) 2.Double signal(few) 3.single signal(very few)

Figure.11

Chart 3

The recombination mechanism has it proof that the existence of type c plasmid was confirmed by reverse sequencing result (The sequence of some expressed genes driven by pEF1a are short enough to be covered and passed through by reverse sequencing),and that of type B plasmid was confirmed by double sequencing signal. Such recombination mechanism can also be supported by digestion of XhoI restriction site which exists right between CPPT and the 188-bp key region of pEF1a,and in the downstream of expressed genes.

Figure 11. (A).Hypothetical calculation of the result of digestion of XhoI restriction sites.(B).Agarose gel image of the result of digestion of XhoI restriction sites,and the verification of recombination

Chart 4

According to the sequencing and digestion result, the recombination stopped in the downstream of CPPT cassette，unlike a dsDNA whose recombination will terminate at the end of the strain. Due to variable terminating position of recombination on our plasmid p199-537bp pEF1a, the impact on pEF1a and the expression of the genes driven can be different. However,as the first XhoI restriction site appears right between CPPT cassette and 170bps-key region( The 170bps region ,damage of which may has devastating impact on the expression of downstream genes,is found in almost every type of pEF1a), any recombination mode whose terminating point passes through the end of CPPT cassette will destroy XhoI site, therefore the XhoI digestion test(figure.11 B) is acute of any damaging recombination. The variable termination of recombination on our plasmid has its proof that some of plasmids with a single signal of forward sequencing, turned out to be recombined in XhoI digestion test.

Figure 12. Multiple recombination modes

Solutions 2013.8.1-8.22

As the function of CPPT cassette inside pEF1a remains unclear,Several strategies were adopted to deal with the recombination of p199-537bp plasmid and keep such 537bps-pEF1a intact,but none of them has worked out ideally so far.

Strategy A

Reconstruct all p199-537bp pEF1a and transport them into competent bacteria MDS42 strain whose genes expressing recombinases are completed knocked off, instead of Top10 strain.

Result: Failure in collecting single-clone colony, as the competence level of MDS42 strain is extreme low.

Stategy B

Pick up massive single-clone colonies(Top10) and conduct plasmids extraction

Result: All plasmid in XhoI digestion test is positive in recombination.

Then we cutted the growth time in 37℃ thermostat shaker down to 8h in,as we hypothesized that the recombination process mainly occurred during bacteria growth in LB medium. But results were still the same.

Strategy C

Pick up all colonies in the plate and concentrate them for direct plasmids extraction. The plasmids are then retransported to MDS42 strain, whose single-clone colonies will be selected again for plasmids extraction.

Status: currently still underway.

In such case , with the problem of recombination of 537bps-pEF1a unsolved, we chose to replace it with 188bps-pEF1a which was reported still possessing at least 30% of driving intensity of a full-length version pEF1a.And theoretically, the 188bps-pEF1a won’t be recombination capable.

Currently we have successfully constructed p199-188bps pEF1a-tTA-PGK-BSD.